1-(5-tert-butyl-2-phenyl-2H-pyrazol-3-yl)-3-[2-fluoro-4-(1-methyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-7-yloxy)-phenyl]-urea and related compounds and their use in therapy

ABSTRACT

The present invention pertains generally to the field of therapeutic compounds, and more specifically to certain compounds of the following formula (for convenience, collectively referred to herein as “IP compounds”), which, inter alia, are useful in the treatment of cancer, e.g., cancer characterized by (e.g., driven by) mutant RAS (“mutant RAS cancer”). The present invention also pertains to pharmaceutical compositions comprising such compounds, and the use of such compounds and compositions in the treatment of cancer, e.g., mutant RAS cancer.

RELATED APPLICATION

This application is related to U.S. patent application No. 61/300,085 filed 1 Feb. 2010, the contents of which are incorporated herein by reference in their entirety.

TECHNICAL FIELD

The present invention pertains generally to the field of therapeutic compounds, and more specifically to certain compounds (for convenience, collectively referred to herein as “IP compounds”), which, inter alia, are useful in the treatment of cancer, e.g., cancer characterised by (e.g., driven by) mutant RAS (“mutant RAS cancer”). The present invention also pertains to pharmaceutical compositions comprising such compounds, and the use of such compounds and compositions in the treatment of cancer, e.g., mutant RAS cancer.

BACKGROUND

A number of patents and publications are cited herein in order to more fully describe and disclose the invention and the state of the art to which the invention pertains. Each of these references is incorporated herein by reference in its entirety into the present disclosure, to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference.

Throughout this specification, including the claims which follow, unless the context requires otherwise, the word “comprise,” and variations such as “comprises” and “comprising,” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a pharmaceutical carrier” includes mixtures of two or more such carriers, and the like.

Ranges are often expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about,” it will be understood that the particular value forms another embodiment.

This disclosure includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

Cancer and RAS

RAS proteins are small-guanine nucleotide binding proteins that are downstream of growth factor, cytokine and hormone receptors. These cell surface receptors activate proteins called guanine-nucleotide exchange factors (GNEFs), which replace GDP for GTP on RAS proteins, stimulating RAS activation. Other proteins called GTPase-activating proteins (GAPs) stimulate the intrinsic GTPase activity of RAS, thereby promoting GTP hydrolysis and returning RAS to its inactive GDP-bound state. Activated RAS binds to several effector proteins, including phosphoinositide 3-kinase (PI3K), the RAF family of protein kinases, and the Ral guanine-nucleotide exchange factor. These effectors in turn regulate the activity of the signalling pathways that control cell proliferation, senescence, survival and differentiation. There are three RAS genes in mammals called HRAS, KRAS and NRAS and they serve overlapping but non-conserved functions.

RAS proteins are also important in cancer. 20-30% of human tumours harbour somatic gain-of-function mutations in one of the RAS genes. Most commonly these involve the codons for glycine 12 (G12), glycine 13 (G13) and glutamine 61 (Q61) and these mutations impair, through different mechanisms, the GAP-stimulated intrinsic GTPase activity of RAS, trapping it in the active GTP-bound state and allowing it to promote tumorigenesis. See, e.g., Downward, 2003; Young et al., 2009; and Bos, 1989.

TABLE 1 Frequency of RAS Mutatations in Different Types of Cancers Tumour Type Frequency Pancreas 90% Thyroid (Undifferentiated papillary) 60% Thyroid (Follicular) 55% Colorectal 45% Seminoma 45% Myelodysplastic syndrome (MDS) 40% Lung adenocarcinoma (non-small-cell) 35% Liver 30% Acute myelogenous leukemia (AML) 30% Melanoma 15% Bladder 10% Kidney 10% RAS and BRAF

Active RAS proteins activate several downstream effectors, including the proteins of the RAF family. There are three RAF proteins, ARAF, BRAF and CRAF. Activated RAF phosphorylates and activates a second protein kinase called MEK, which then phosphorylates and activates a third protein kinase called ERK. ERK phosphorylates a multitude of cytosolic and nuclear substrates, thereby regulating cell processes such as proliferation, survival, differentiation and senescence.

BRAF is important in cancer, because it is mutated in about 2% of human cancers, particularly in melanoma (43% of cases), thyroid (45%), ovarian (10%), and colorectal (13%) cancers. In contrast, ARAF and CRAF mutations are very rare in human cancer. Notably, however, in cancer cells, oncogenic RAS does not signal through BRAF, but instead signals exclusively through CRAE to activate MEK.

Over 100 different mutations have been described in BRAF in cancer, but a single mutation (a glutamic acid substitution for the valine at position 600) accounts for about 90% of the mutations that occur. This mutant activates BRAF 500-fold, and allows it to stimulate constitutive ERK and NEkB signalling, stimulating survival and proliferation. Consequently, ^(V600E)BRAF can transform cells such as fibroblasts and melanocytes. Inhibition of ^(V600E)BRAF in cancer cells inhibits cell proliferation and induces apoptosis in vitro, and in vivo it suppresses tumor cell growth, validating ^(V600E)BRAF as a therapeutic target.

In the vast majority of cancers, BRAF and RAS mutations are mutually exclusive. This provides genetic evidence to suggest that these proteins are on the same pathway and that they drive the same processes in cancer cells. However, there are clear differences between oncogenic BRAF and oncogenic RAS functions in cancer cells. First, RAS activates several pathways, whereas BRAF is only known to activate the MEK/ERK pathway. As a consequence, BRAF mutant cells are more dependent on MEK/ERK signalling and so are considerably more sensitive to BRAF or MEK inhibitors than cell in which RAS is mutated. See, e.g., Garnett et al., 2004; Wellbrock et al., 2004; Gray-Schopfer et al., 2007; Solit et al., 2006.

Related Compounds

Niculescu-Duvaz et al., 2006 (WO 2006/043090 A1), describes the following classes of compounds at pages 41 and 43 therein. The compounds are described as BRAF inhibitors useful for the treatment of cancer, especially mutant BRAF cancer.

Additionally, Niculescu-Duvaz et al., 2006 provides the following examples:

Niculescu-Duvaz et al., 2007 (WO 2007/125330 A1), describes the following classes of compounds at pages 57 and 58 therein. The compounds are described as BRAF inhibitors useful for the treatment of cancer, especially mutant BRAF cancer.

Additionally, Niculescu-Duvaz et al., 2007 provides the following examples:

The present invention provides alternative compounds, which are characterized by a particular combination of structural motifs, and which provide surprising and unexpected activity (e.g., activity against mutant RAS cancers), for example, as compared to one or more of the structurally-related known compounds.

Although structurally-related compounds are known as BRAF inhibitors, it would not have been predicted that the claimed compounds are active against mutant RAS cancers.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the chemical structure of several compounds of the present invention: AA-01, AA-02, AA-03, and AA-04.

FIG. 2 shows the chemical structure of several compounds of the present invention: BB-01 and BB-02.

FIG. 3 shows the chemical structure of several comparison compounds: XX-01, XX-02, XX-03, and XX-04.

FIG. 4 is a bar graph showing the ratio of the GI₅₀ for each of a range of cell lines to the GI₅₀ for the WM266.4 cell line for compound AA-01. The range of cell lines are (a) a panel of mutant BRAF (mutBRAF) cell lines: WM266.4, A375M, UACC62; (b) a panel of mutant RAS (mutRAS) cell lines: SW620, HCT116, and WM1361; and (c) a panel of wild type BRAF and RAS (wtBRAFT/RAS) cell lines: SKMEL23. KM12, and BT474.

FIG. 5 is a bar graph showing the ratio of the GI₅₀ for each of a range of cell lines to the GI₅₀ for the WM266.4 cell line for compound BB-01. The ranges of cell lines are as for FIG. 4.

FIG. 6 is a bar graph showing the ratio of the GI₅₀ for each of a range of cell lines to the GI₅₀ for the WM266.4 cell line for compound BB-02. The ranges of cell lines are as for FIG. 4.

FIG. 7 is a bar graph showing the ratio of the GI₅₀ for each of a range of cell lines to the GI₅₀ for the WM266.4 cell line for comparison compound XX-01. The ranges of cell lines are as for FIG. 4.

FIG. 8 is a bar graph showing the ratio of the GI₅₀ for each of a range of cell lines to the GI₅₀ for the WM266.4 cell line for comparison compound XX-02. The ranges of cell lines are as for FIG. 4.

FIG. 9 is a bar graph showing the ratio of the GI₅₀ for each of a range of cell lines to the GI₅₀ for the WM266.4 cell line for comparison compound XX-03. The ranges of cell lines are as for FIG. 4.

FIG. 10 is a bar graph showing the ratio of the GI₅₀ for each of a range of cell lines to the GI₅₀ for the WM266.4 cell line for comparison compound XX-04. The ranges of cell lines are as for FIG. 4.

FIG. 11 is a graph of relative tumour volume as a function of time (days) for mouse xenografts of the mutant RAS cell line SW620, for treatment with compound AA-01 and for controls.

FIG. 12 is a graph of relative tumour volume as a function of time (days) for mouse xenografts of the mutant RAS cell line SW620, for treatment with compound BB-02 and for controls.

FIG. 13 is a graph of relative tumour volume as a function of time (days) for mouse xenografts of the mutant BRAF cell line A375, for treatment with compound AA-01 and for controls.

FIG. 14 is a graph of relative tumour volume as a function of time (days) for mouse xenografts of the mutant BRAF cell line A375, for treatment with compound BB-02 and for controls.

FIG. 15 is a graph of relative tumour volume as a function of time (days) for mouse xenografts of the mutant RAS cell line SW620, for treatment with comparison compound XX-01 and for controls.

FIG. 16 is a graph of relative tumour volume as a function of time (days) for mouse xenografts of the mutant RAS cell line SW620, for treatment with comparison compound XX-02 and for controls.

FIG. 17 is a graph of relative tumour volume as a function of time (days) for mouse xenografts of the mutant RAS cell line SW620, for treatment with comparison compound XX-03 and for controls.

FIG. 18 is a graph of relative tumour volume as a function of time (days) for mouse xenografts of the mutant RAS cell line SW620, for treatment with comparison compound XX-04 and for controls.

FIG. 19 is a graph of relative tumour volume as a function of time (days) for mouse xenografts of the mutant BRAF cell line A375, for treatment with comparison compound XX-02 and for controls.

SUMMARY OF THE INVENTION

One aspect of the invention pertains to certain compounds (for convenience, collectively referred to herein as “IP compounds”), as described herein.

Another aspect of the invention pertains to a composition (e.g., a pharmaceutical composition) comprising an IP compound, as described herein, and a pharmaceutically acceptable carrier or diluent.

Another aspect of the invention pertains to method of preparing a composition (e.g., a pharmaceutical composition) comprising the step of admixing an IP compound, as described herein, and a pharmaceutically acceptable carrier or diluent.

Another aspect of the present invention pertains to a method of treatment comprising administering to a subject in need of treatment a therapeutically-effective amount of an IP compound, as described herein, preferably in the form of a pharmaceutical composition.

Another aspect of the present invention pertains to an IP compound as described herein for use in a method of treatment of the human or animal body by therapy.

Another aspect of the present invention pertains to use of an IP compound, as described herein, in the manufacture of a medicament for use in treatment.

In one embodiment, the treatment is treatment of cancer.

In one embodiment, the cancer is solid tumour cancer.

In one embodiment, the cancer is pancreatic cancer; thyroid (e.g., follicular; undifferentiated papillary) cancer; colorectal cancer; seminoma; myelodysplastic syndrome (MDS); lung cancer (e.g., lung adenocarcinoma); liver cancer; leukemia (e.g., acute myelogenous leukemia (AML)); melanoma; bladder cancer; kidney cancer; breast cancer, ovarian cancer, bile duct cancer, or glioma.

In one embodiment, the cancer is pancreatic cancer.

In one embodiment, the cancer is thyroid cancer.

In one embodiment, the cancer is colorectal cancer.

In one embodiment, the cancer is seminoma.

In one embodiment, the cancer is myelodysplastic syndrome (MDS).

In one embodiment, the cancer is lung cancer.

In one embodiment, the cancer is lung adenocarcinoma.

In one embodiment, the cancer is liver cancer.

In one embodiment, the cancer is leukemia.

In one embodiment, the cancer is acute myelogenous leukemia (AML).

In one embodiment, the cancer is melanoma.

In one embodiment, the cancer is bladder cancer.

In one embodiment, the cancer is kidney cancer.

In one embodiment, the cancer is breast cancer.

In one embodiment, the cancer is ovarian cancer.

In one embodiment, the cancer is bile duct cancer.

In one embodiment, the cancer is glioma.

In one embodiment, the cancer is mutant RAS cancer (e.g., mutant RAS pancreatic cancer, etc.).

In one embodiment, the cancer is characterised by, or further characterised by, cancer stem cells.

In one embodiment, the treatment further comprises treatment with one or more additional therapeutic agents or therapies, for example, one or more additional a cancer agents or therapies.

Another aspect of the present invention pertains to a kit comprising (a) an IP compound, as described herein, preferably provided as a pharmaceutical composition and in a suitable container and/or with suitable packaging; and (b) instructions for use, for example, written instructions on how to administer the compound.

Another aspect of the present invention pertains to an IP compound obtainable by a method of synthesis as described herein, or a method comprising a method of synthesis as described herein.

Another aspect of the present invention pertains to an IP compound obtained by a method of synthesis as described herein, or a method comprising a method of synthesis as described herein.

Another aspect of the present invention pertains to novel intermediates, as described herein, which are suitable for use in the methods of synthesis described herein.

Another aspect of the present invention pertains to the use of such novel intermediates, as described herein, in the methods of synthesis described herein.

As will be appreciated by one of skill in the art, features and preferred embodiments of one aspect of the invention will also pertain to other aspect of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Compounds

The present relates to certain compounds which are structurally related to the following compounds:

In contrast to known compounds, the compounds of the present invention are characterized by a particular combination of structural motifs, specifically:

(A) a 1-methyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-7-yloxy motif:

(B) a linking 2-fluoro-phen-1,4-di-yl moiety or a linking naphth-1,4-di-yl motif:

(C) a 1-(5-tert-butyl-2-phenyl-2H-pyrazol-3-yl)-ureyl motif, where the phenyl group optionally bears a meta- or para-substituent:

None of the known compounds have all three of these motifs. Furthermore, comparison studies with compounds (including known compounds) demonstrate that, surprisingly and unexpectedly, compounds having all three of the motifs (i.e., the claimed compounds) have substantially better activity against mutant RAS cancers, than comparison compounds which lack one or more of these motifs.

Thus, one aspect of the present invention pertains to compounds of the following formula, and pharmaceutically acceptable salts, hydrates, and solvates thereof (collectively referred to herein as “IP compounds”):

wherein -J- is independently:

and wherein —R is independently —H, -Me, —F, —Cl, —Br, or —I;

and wherein —R is positioned meta- or para- on the phenyl ring.

In one embodiment, the compound is selected from compounds of the following formula, and pharmaceutically acceptable salts, hydrates, and solvates thereof, wherein —R^(A) is independently —H, —F, —Cl, —Br, or —I, and wherein —R^(A) is positioned meta- or para- on the phenyl ring:

In one embodiment, the compound is selected from compounds of the following formula, and pharmaceutically acceptable salts, hydrates, and solvates thereof, wherein —R^(A) is independently —H, —F, —Cl, —Br, or —I:

In one embodiment, the compound is selected from compounds of the following formula, and pharmaceutically acceptable salts, hydrates, and solvates thereof, wherein —R^(A) is independently —H, -Me, —F, —Cl, —Br, or —I:

In one embodiment, —R^(A) is independently —H, -Me, —F, or —Cl.

In one embodiment, —R^(A) is independently —H or -Me.

In one embodiment, —R^(A) is independently —H.

In one embodiment, —R^(A) is independently -Me.

In one embodiment, —R^(A) is independently —F or —Cl.

In one embodiment, —R^(A) is independently —F.

In one embodiment, —R^(A) is independently —Cl.

In one embodiment, the compound is selected from the following compound (i.e., AA-01), and pharmaceutically acceptable salts, hydrates, and solvates thereof:

In one embodiment, the compound is selected from the following compound (i.e., AA-02), and pharmaceutically acceptable salts, hydrates, and solvates thereof:

In one embodiment, the compound is selected from the following compound (i.e., AA-03), and pharmaceutically acceptable salts, hydrates, and solvates thereof:

In one embodiment, the compound is selected from the following compound (i.e., AA-04), and pharmaceutically acceptable salts, hydrates, and solvates thereof:

In one embodiment, the compound is selected from compounds of the following formula, and pharmaceutically acceptable salts, hydrates, and solvates thereof, wherein —R⁸ is independently —H, -Me, —F, —Cl, —Br, or —I, and wherein —R⁸ is positioned meta- or para- on the phenyl ring:

In one embodiment, the compound is selected from compounds of the following formula, and pharmaceutically acceptable salts, hydrates, and solvates thereof, wherein —R⁸ is independently —H, -Me, —F, —Cl, —Br, or —I:

and pharmaceutically acceptable salts, hydrates, and solvates thereof, wherein —R^(B) is independently —H, -Me, —F, —Cl, —Br, or —I:

In one embodiment, —R^(B) is independently —H, -Me, —F, or —Cl.

In one embodiment, —R^(B) is independently —H or -Me.

In one embodiment, —R^(B) is independently —H.

In one embodiment, —R^(B) is independently -Me.

In one embodiment, —R^(B) is independently —F or —Cl.

In one embodiment, —R^(B) is independently —F.

In one embodiment, —R^(B) is independently —Cl.

In one embodiment, the compound is selected from the following compound (i.e., BB-01), and pharmaceutically acceptable salts, hydrates, and solvates thereof:

In one embodiment, the compound is selected from the following compound (i.e., BB-02), and pharmaceutically acceptable salts, hydrates, and solvates thereof:

Combinations

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the chemical groups represented by the variables (e.g., -J-, —R, —R^(A), —R^(B), etc.) are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed, to the extent that such combinations embrace compounds that are stable compounds (i.e., compounds that can be isolated, characterised, and tested for biological activity). In addition, all sub-combinations of the chemical groups listed in the embodiments describing such variables are also specifically embraced by the present invention and are disclosed herein just as it each and every such sub-combination of chemical groups was individually and explicitly disclosed herein.

Substantially Purified Forms

One aspect of the present invention pertains to IP compounds, as described herein, in substantially purified form and/or in a form substantially free from contaminants.

In one embodiment, the compound is in substantially purified form and/or in a form substantially free from contaminants.

In one embodiment, the compound is in a substantially purified form with a purity of least 50% by weight, e.g., at least 60% by weight, e.g., at least 70% by weight, e.g., at least 80% by weight, e.g., at least 90% by weight, e.g., at least 95% by weight, e.g., at least 97% by weight, e.g., at least 98% by weight, e.g., at least 99% by weight.

Unless specified, the substantially purified form refers to the compound in any stereoisomeric form. For example, in one embodiment, the substantially purified form refers to a mixture of stereoisomers, i.e., purified with respect to other compounds. In one embodiment, the substantially purified form refers to one stereoisomer.

In one embodiment, the compound is in a form substantially free from contaminants wherein the contaminants represent no more than 50% by weight, e.g., no more than 40% by weight, e.g., no more than 30% by weight, e.g., no more than 20% by weight, e.g., no more than 10% by weight, e.g., no more than 5% by weight, e.g., no more than 3% by weight, e.g., no more than 2% by weight, e.g., no more than 1% by weight.

Unless specified, the contaminants refer to other compounds, that is, other than stereoisomers. In one embodiment, the contaminants refer to other compounds and other stereoisomers.

Isomers

Certain compounds may exist in one or more particular geometric, optical, enantiomeric, diasteriomeric, epimeric, atropic, stereoisomeric, tautomeric, conformational, or anomeric forms, including but not limited to, cis- and trans-forms; E- and Z-forms; c-, t-, and r-forms; endo- and exo-forms; R—, S—, and meso-forms; D- and L-forms; d- and l-forms; (+) and (−) forms; keto-, enol-, and enolate-forms; syn- and anti-forms; synclinal- and anticlinal-forms; α- and β-forms; axial and equatorial forms; boat-, chair-, twist-, envelope-, and halfchair-forms; and combinations thereof, hereinafter collectively referred to as “isomers” (or “isomeric forms”).

Note that specifically excluded from the term “isomers,” as used herein, are structural (or constitutional) isomers (i.e., isomers which differ in the connections between atoms rather than merely by the position of atoms in space). For example, a reference to a tert-butyl group, —C(CH₃)₃, is not to be construed as a reference to its structural isomer, iso-butyl, —CH₂CH(CH₃)₂. Similarly, a reference to para-chlorophenyl is not to be construed as a reference to its structural isomer, meta-chlorophenyl.

Note that specifically included in the term “isomer” are compounds with one or more isotopic substitutions. For example, H may be in any isotopic form, including ¹H, ²H (D), and ³H (T); C may be in any isotopic form, including ¹²C, ¹³C, and ¹⁴C; O may be in any isotopic form, including ¹⁶O and ¹⁸O; and the like.

Unless otherwise specified, a reference to a particular compound includes all such isomeric forms, including mixtures thereof.

Salts

It may be convenient or desirable to prepare, purify, and/or handle a corresponding salt of the compound, for example, a pharmaceutically-acceptable salt. Examples of pharmaceutically acceptable salts are discussed in Berge et al., 1977, “Pharmaceutically Acceptable Salts,” J. Pharm. Sci., Vol. 66, pp. 1-19.

For example, if the compound is cationic, or has a functional group which may be cationic (e.g., —NH— may be —NH₂ ⁺—), then a salt may be formed with a suitable anion. Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric, nitrous, phosphoric, and phosphorous.

Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetyoxybenzoic, acetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric, edetic, ethanedisulfonic, ethanesulfonic, fumaric, glucheptonic, gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalene carboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic, methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic, phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic, succinic, sulfanilic, tartaric, toluenesulfonic, and valeric. Examples of suitable polymeric organic anions include, but are not limited to, those derived from the following polymeric acids: tannic acid, carboxymethyl cellulose.

Unless otherwise specified, a reference to a particular compound also includes salt forms thereof.

Hydrates and Solvates

It may be convenient or desirable to prepare, purify, and/or handle a corresponding solvate of the compound. The term “solvate” is used herein in the conventional sense to refer to a complex of solute (e.g., compound, salt of compound) and solvent. If the solvent is water, the solvate may be conveniently referred to as a hydrate, for example, a mono-hydrate, a di-hydrate, a tri-hydrate, etc.

Unless otherwise specified, a reference to a particular compound also includes solvate and hydrate forms thereof.

Chemical Synthesis

Methods for the chemical synthesis of compounds of the present invention are described herein. These and/or other well known methods may be modified and/or adapted in known ways in order to facilitate the synthesis of additional compounds within the scope of the present invention.

Descriptions of general laboratory methods and procedures, useful for the preparation of the compounds described herein, are provided in Vogel's Textbook of Practical Organic Chemistry 5th Edition, 1989, (Editors: Furniss, Hannaford, Smith, and Tatchell) (published by Longmann, UK).

Methods for the synthesis of pyridine compounds in particular are described in Heterocyclic Chemistry, 3rd Edition, 1998, (Editors: Joule, Mills, and Smith) (published by Chapman & Hall, UK).

The IP compounds described herein may be prepared via intermediates (2). These intermediates may be prepared from commercially available starting material, 2-amino-3-nitro-4-chloropyridine (1), and 3-fluoro-4-aminophenol (R¹ is —H and R² is —F) or 4-amino-1-naphthol (R¹ and R₂ together are —CH═CH—). Intermediates (2) are then protected selectively at the amino group, for example as a BOC carbamate, to afford intermediates (3).

The intermediates (3) can also be obtained directly from 2-amino-3-nitro-4-chloropyridine (1) and N-BOC-protected 3-fluoro-4-aminophenol or N-BOC-protected 4-amino-1-naphthol.

The nitro group of the protected intermediates (3) may be reduced to an amino group with Pd/C and ammonium formate or hydrogen, or with NiCl₂ and NaBH₄, to give the diamino intermediates (4).

The intermediates (8), alkylated at N3 (by respect to the pyridine ring), may be prepared from intermediates (4). The more nucleophilic 3-amino group on intermediates (4) is converted to ethyl carbamate, to afford intermediates (5), and the BOC group is removed with TFA to afford intermediates (6). Deprotonation of the acidic carbamate proton with NaH gives an anion on N3 that is alkylated to afford the intermediates (7). Cyclisation of intermediates (7), in the presence of base, gives the corresponding intermediates (8).

The intermediates (8) are reacted with 3-tert-butyl-5-isocyanato-1-aryl-1H-pyrazoles to afford the corresponding ureas. The respective isocyanates can be obtained either by the reaction of amines with phosgene, triphosgene or their derivatives, or by conversion of the corresponding carboxylic acids to acyl azides with, for example, diphenyl phosphoryl azide, followed by Curtius rearrangement. The aryl group on the pyrazole may be, for example, unsubstituted or substituted (e.g., meta or para-substituted) with an alkyl group (e.g., -Me) or a halogen atom (e.g., —F, —Cl, —Br, —I).

Compositions

One aspect of the present invention pertains to a composition (e.g., a pharmaceutical composition) comprising an IP compound, as described herein, and a pharmaceutically acceptable carrier, diluent, or excipient.

In one embodiment, the composition further comprises one or more (e.g., 1, 2, 3, 4) additional therapeutic agents, as described herein.

Another aspect of the present invention pertains to a method of preparing a composition (e.g., a pharmaceutical composition) comprising admixing an IP compound, as described herein, and a pharmaceutically acceptable carrier, diluent, or excipient.

Another aspect of the present invention pertains to a method of preparing a composition (e.g., a pharmaceutical composition) comprising admixing an IP compound, as described herein; one or more (e.g., 1, 2, 3, 4) additional therapeutic agents, as described herein; and a pharmaceutically acceptable carrier, diluent, or excipient.

Uses

The compounds described herein are useful, for example, in the treatment of cancer, for example, mutant RAS cancer.

For the avoidance of doubt, the term “mutant RAS cancer” is used herein to refer to cancer that is characterised by (e.g., driven by) mutant RAS, for example, by one or more mutations (e.g., gain-of-function mutations) in one of the RAS genes (i.e., HRAS, KRAS, and NRAS). As discussed herein, the most common RAS mutations involve codons for one or more of glycine 12 (G12), glycine 13 (G13) and glutamine 61 (061).

Use in Methods of Therapy

Another aspect of the present invention pertains to an IP compound, as described herein, for use in a method of treatment of the human or animal body by therapy.

Another aspect of the present invention pertains to an IP compound, as described herein, in combination with one or more (e.g., 1, 2, 3, 4) additional therapeutic agents, as described herein, for use in a method of treatment of the human or animal body by therapy.

Use in the Manufacture of Medicaments

Another aspect of the present invention pertains to use of an IP compound, as described herein, in the manufacture of a medicament for use in treatment.

In one embodiment, the medicament comprises the IP compound.

Another aspect of the present invention pertains to use of an IP compound, as described herein, and one or more (e.g., 1, 2, 3, 4) additional therapeutic agents, as described herein, in the manufacture of a medicament for use in treatment.

In one embodiment, the medicament comprises the IP compound and the one or more (e.g., 1, 2, 3, 4) additional therapeutic agents.

Methods of Treatment

Another aspect of the present invention pertains to a method of treatment comprising administering to a patient in need of treatment a therapeutically effective amount of an IP compound, as described herein, preferably in the form of a pharmaceutical composition.

Another aspect of the present invention pertains to a method of treatment comprising administering to a patient in need of treatment a therapeutically effective amount of an IP compound, as described herein, preferably in the form of a pharmaceutical composition, and one or more (e.g., 1, 2, 3, 4) additional therapeutic agents, as described herein, preferably in the form of a pharmaceutical composition.

Conditions Treated

In one embodiment, the treatment is treatment of cancer.

In one embodiment, the cancer is lung cancer, small cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, stomach cancer, bowel cancer, colorectal cancer, thyroid cancer, breast cancer, ovarian cancer, endometrial cancer, prostate cancer, testicular cancer, liver cancer, kidney cancer, renal cell carcinoma, bladder cancer, pancreatic cancer, brain cancer, glioma, sarcoma, osteosarcoma, bone cancer, nasopharyngeal cancer (e.g., head cancer, neck cancer), skin cancer, squamous cancer, Kaposi's sarcoma, melanoma, malignant melanoma, lymphoma, or leukemia.

In one embodiment, the cancer is:

-   -   a carcinoma, for example a carcinoma of the bladder, breast,         colon/rectum (e.g., colorectal carcinomas such as colon         adenocarcinoma and colon adenoma), kidney, epidermal, liver,         lung (e.g., adenocarcinoma, small cell lung cancer and non-small         cell lung carcinomas), oesophagus, gall bladder, ovary, pancreas         (e.g., exocrine pancreatic carcinoma), stomach, cervix, thyroid,         prostate, skin (e.g., squamous cell carcinoma);     -   a hematopoietic tumour of lymphoid lineage, for example         leukemia, acute lymphocytic leukemia, B-cell lymphoma, T-cell         lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell         lymphoma, or Burkett's lymphoma;     -   a hematopoietic tumor of myeloid lineage, for example acute and         chronic myelogenous leukemias, myelodysplastic syndrome, or         promyelocytic leukemia;     -   a tumour of mesenchymal origin, for example fibrosarcoma or         habdomyosarcoma;     -   a tumor of the central or peripheral nervous system, for example         astrocytoma, neuroblastoma, glioma or schwannoma;     -   melanoma; seminoma; teratocarcinoma; osteosarcoma; xenoderoma         pigmentoum; keratoctanthoma; thyroid follicular cancer; or         Kaposi's sarcoma.

In one embodiment, the cancer is solid tumour cancer.

In one embodiment, the cancer is pancreatic cancer; thyroid (e.g., follicular; undifferentiated papillary) cancer; colorectal cancer; seminoma; myelodysplastic syndrome (MDS); lung cancer (e.g., lung adenocarcinoma); liver cancer; leukemia (e.g., acute myelogenous leukemia (AML)); melanoma; bladder cancer; kidney cancer; breast cancer, ovarian cancer, bile duct cancer, or glioma.

In one embodiment, the cancer is pancreatic cancer.

In one embodiment, the cancer is thyroid cancer.

In one embodiment, the cancer is colorectal cancer.

In one embodiment, the cancer is seminoma.

In one embodiment, the cancer is myelodysplastic syndrome (MDS).

In one embodiment, the cancer is lung cancer.

In one embodiment, the cancer is lung adenocarcinoma.

In one embodiment, the cancer is liver cancer.

In one embodiment, the cancer is leukemia.

In one embodiment, the cancer is acute myelogenous leukemia (AML).

In one embodiment, the cancer is melanoma.

In one embodiment, the cancer is bladder cancer.

In one embodiment, the cancer is kidney cancer.

In one embodiment, the cancer is breast cancer.

In one embodiment, the cancer is ovarian cancer.

In one embodiment, the cancer is bile duct cancer.

In one embodiment, the cancer is glioma.

In one embodiment, the cancer is mutant RAS cancer (e.g., mutant RAS pancreatic cancer, etc.).

In one embodiment, the cancer is characterised by, or further characterised by, cancer stem cells.

The anti-cancer effect may arise through one or more mechanisms, including but not limited to, the regulation of cell proliferation, the inhibition of cell cycle progression, the inhibition of angiogenesis (the formation of new blood vessels), the inhibition of metastasis (the spread of a tumour from its origin), the inhibition of cell migration (the spread of cancer cells to other parts of the body), the inhibition of invasion (the spread of tumour cells into neighbouring normal structures), or the promotion of apoptosis (programmed cell death). The compounds of the present invention may be used in the treatment of the cancers described herein, independent of the mechanisms discussed herein.

Treatment

The term “treatment,” as used herein in the context of treating a condition, pertains generally to treatment and therapy, whether of a human or an animal (e.g., in veterinary applications), in which some desired therapeutic effect is achieved, for example, the inhibition of the progress of the condition, and includes a reduction in the rate of progress, a halt in the rate of progress, alleviatiation of symptoms of the condition, amelioration of the condition, and cure of the condition. Treatment as a prophylactic measure (i.e., prophylaxis) is also included. For example, use with patients who have not yet developed the condition, but who are at risk of developing the condition, is encompassed by the term “treatment.”

For example, treatment includes the prophylaxis of cancer, reducing the incidence of cancer, reducing the severity of cancer, alleviating the symptoms of cancer, etc.

The term “therapeutically-effective amount,” as used herein, pertains to that amount of a compound, or a material, composition or dosage form comprising a compound, which is effective for producing some desired therapeutic effect, commensurate with a reasonable benefit/risk ratio, when administered in accordance with a desired treatment regimen.

Combination Therapies

The term “treatment” includes combination treatments and therapies, in which two or more treatments or therapies are combined, for example, sequentially or simultaneously. For example, the compounds described herein may also be used in combination therapies, e.g., in conjunction with other agents, for example, cytotoxic agents, anticancer agents, etc. Examples of treatments and therapies include, but are not limited to, chemotherapy (the administration of active agents, including, e.g., drugs, antibodies (e.g., as in immunotherapy), prodrugs (e.g., as in photodynamic therapy, GDEPT, ADEPT, etc.); surgery; radiation therapy; photodynamic therapy; gene therapy; and controlled diets.

For example, it may be beneficial to combine treatment with a compound as described herein with one or more other (e.g., 1, 2, 3, 4) agents or therapies that regulates cell growth or survival or differentiation via a different mechanism, thus treating several characteristic features of cancer development.

One aspect of the present invention pertains to a compound as described herein, in combination with one or more additional therapeutic agents, as described below.

The particular combination would be at the discretion of the physician who would select dosages using his common general knowledge and dosing regimens known to a skilled practitioner.

The agents (i.e., the compound described herein, plus one or more other agents) may be administered simultaneously or sequentially, and may be administered in individually varying dose schedules and via different routes. For example, when administered sequentially, the agents can be administered at closely spaced intervals (e.g., over a period of 5-10 minutes) or at longer intervals (e.g., 1, 2, 3, 4 or more hours apart, or even longer periods apart where required), the precise dosage regimen being commensurate with the properties of the therapeutic agent(s).

The agents (i.e., the compound described here, plus one or more other agents) may be formulated together in a single dosage form, or alternatively, the individual agents may be formulated separately and presented together in the form of a kit, optionally with instructions for their use.

Other Uses

The IP compounds described herein may also be used as part of an in vitro assay, for example, in order to determine whether a candidate host is likely to benefit from treatment with the compound in question.

The IP compounds described herein may also be used as a standard, for example, in an assay, in order to identify other compounds, other anti-cancer agents, etc.

Kits

One aspect of the invention pertains to a kit comprising (a) an IP compound as described herein, or a composition comprising an IP compound as described herein, e.g., preferably provided in a suitable container and/or with suitable packaging; and (b) instructions for use, e.g., written instructions on how to administer the compound or composition.

In one embodiment, the kit further comprises one or more (e.g., 1, 2, 3, 4) additional therapeutic agents, as described herein.

The written instructions may also include a list of indications for which the active ingredient is a suitable treatment.

Route Administration

The IP compound or pharmaceutical composition comprising the IP compound may be administered to a subject by ally convenient route of administration, whether systemically/peripherally or topically (i.e., at the site of desired action).

Routes of administration include, but are not limited to, oral (e.g., by ingestion); buccal; sublingual; transdermal (including, e.g., by a patch, plaster, etc.); transmucosal (including, e.g., by a patch, plaster, etc.); intranasal (e.g., by nasal spray, drops or from an atomiser or dry powder delivery device); ocular (e.g., by eyedrops); pulmonary (e.g., by inhalation or insufflation therapy using, e.g., an aerosol, e.g., through the mouth or nose); rectal (e.g., by suppository or enema); vaginal (e.g., by pessary); parenteral, for example, by injection, including subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, and intrasternal; by implant of a depot or reservoir, for example, subcutaneously or intramuscularly.

The Subject/Patient

The subject/patient may be a chordate, a vertebrate, a mammal, a placental mammal, a marsupial (e.g., kangaroo, wombat), a rodent (e.g., a guinea pig, a hamster, a rat, a mouse), murine (e.g., a mouse), a lagomorph (e.g., a rabbit), avian (e.g., a bird), canine (e.g., a dog), feline (e.g., a cat), equine (e.g., a horse), porcine (e.g., a pig), ovine (e.g., a sheep), bovine (e.g., a cow), a primate, simian (e.g., a monkey or ape), a monkey (e.g., marmoset, baboon), an ape (e.g., gorilla, chimpanzee, orangutang, gibbon), or a human.

Furthermore, the subject/patient may be any of its forms of development, for example, a foetus.

In one preferred embodiment, the subject/patient is a human.

Formulations

While it is possible for the IP compound to be administered alone, it is preferable to present it as a pharmaceutical formulation (e.g., composition, preparation, medicament) comprising at least one IP compound, as described herein, together with one or more other pharmaceutically acceptable ingredients well known to those skilled in the art, including, but not limited to, pharmaceutically acceptable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, anti-oxidants, lubricants, stabilisers, solubilisers, surfactants (e.g., wetting agents), masking agents, colouring agents, flavouring agents, and sweetening agents. The formulation may further comprise other active agents, for example, other therapeutic or prophylactic agents.

Thus, the present invention further provides pharmaceutical compositions, as defined above, and methods of making a pharmaceutical composition comprising admixing at least one IP compound, as described herein, together with one or more other pharmaceutically acceptable ingredients well known to those skilled in the art, e.g., carriers, diluents, excipients, etc. If formulated as discrete units (e.g., tablets, etc.), each unit contains a predetermined amount (dosage) of the compound.

The term “pharmaceutically acceptable,” as used herein, pertains to compounds, ingredients, materials, compositions, dosage forms, etc., which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of the subject in question (e.g., human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Each carrier, diluent, excipient, etc. must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation.

Suitable carriers, diluents, excipients, etc. can be found in standard pharmaceutical texts, for example, Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Company, Easton, Pa., 1990; and Handbook of Pharmaceutical Excipients, 5th edition, 2005.

The formulations may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the compound with a carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the compound with carriers (e.g., liquid carriers, finely divided solid carrier, etc.), and then shaping the product, if necessary.

The formulation may be prepared to provide for rapid or slow release; immediate, delayed, timed, or sustained release; or a combination thereof.

Formulations may suitably be in the form of liquids, solutions (e.g., aqueous, non-aqueous), suspensions (e.g., aqueous, non-aqueous), emulsions (e.g., oil-in-water, water-in-oil), elixirs, syrups, electuaries, mouthwashes, drops, tablets (including, e.g., coated tablets), granules, powders, losenges, pastilles, capsules (including, e.g., hard and soft gelatin capsules), cachets, pills, ampoules, boluses, suppositories, pessaries, tinctures, gels, pastes, ointments, creams, lotions, oils, foams, sprays, mists, or aerosols.

Formulations may suitably be provided as a patch, adhesive plaster, bandage, dressing, or the like which is impregnated with one or more compounds and optionally one or more other pharmaceutically acceptable ingredients, including, for example, penetration, permeation, and absorption enhancers. Formulations may also suitably be provided in the form of a depot or reservoir.

The compound may be dissolved in, suspended in, or admixed with one or more other pharmaceutically acceptable ingredients. The compound may be presented in a liposome or other microparticulate which is designed to target the compound, for example, to blood components or one or more organs.

Formulations suitable for oral administration (e.g., by ingestion) include liquids, solutions (e.g., aqueous, non-aqueous), suspensions (e.g., aqueous, non-aqueous), emulsions (e.g., oil-in-water, water-in-oil), elixirs, syrups, electuaries, tablets, granules, powders, capsules, cachets, pills, ampoules, boluses.

Formulations suitable for buccal administration include mouthwashes, losenges, pastilles, as well as patches, adhesive plasters, depots, and reservoirs. Losenges typically comprise the compound in a flavored basis, usually sucrose and acacia or tragacanth. Pastilles typically comprise the compound in an inert matrix, such as gelatin and glycerin, or sucrose and acacia. Mouthwashes typically comprise the compound in a suitable liquid carrier.

Formulations suitable for sublingual administration include tablets, losenges, pastilles, capsules, and pills.

Formulations suitable for oral transmucosal administration include liquids, solutions (e.g., aqueous, non-aqueous), suspensions (e.g., aqueous, non-aqueous), emulsions (e.g., oil-in-water, water-in-oil), mouthwashes, losenges, pastilles, as well as patches, adhesive plasters, depots, and reservoirs.

Formulations suitable for non-oral transmucosal administration include liquids, solutions (e.g., aqueous, non-aqueous), suspensions (e.g., aqueous, non-aqueous), emulsions (e.g., oil-in-water, water-in-oil), suppositories, pessaries, gels, pastes, ointments, creams, lotions, oils, as well as patches, adhesive plasters, depots, and reservoirs.

Formulations suitable for transdermal administration include gels, pastes, ointments, creams, lotions, and oils, as well as patches, adhesive plasters, bandages, dressings, depots, and reservoirs.

Tablets may be made by conventional means, e.g., compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the compound in a free-flowing form such as a powder or granules, optionally mixed with one or more binders (e.g., povidone, gelatin, acacia, sorbitol, tragacanth, hydroxypropylmethyl cellulose); fillers or diluents (e.g., lactose, microcrystalline cellulose, calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, silica); disintegrants (e.g., sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose); surface-active or dispersing or wetting agents (e.g., sodium lauryl sulfate); preservatives (e.g., methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, sorbic acid); flavours, flavour enhancing agents, and sweeteners. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the compound therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile. Tablets may optionally be provided with a coating, for example, to affect release, for example an enteric coating, to provide release in parts of the gut other than the stomach.

Ointments are typically prepared from the compound and a paraffinic or a water-miscible ointment base.

Creams are typically prepared from the compound and an oil-in-water cream base. If desired, the aqueous phase of the cream base may include, for example, at least about 30% w/w of a polyhydric alcohol, i.e., an alcohol having two or more hydroxyl groups such as propylene glycol, butane-1,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol and mixtures thereof. The topical formulations may desirably include a compound which enhances absorption or penetration of the compound through the skin or other affected areas. Examples of such dermal penetration enhancers include dimethylsulfoxide and related analogues.

Emulsions are typically prepared from the compound and an oily phase, which may optionally comprise merely an emulsifier (otherwise known as an emulgent), or it may comprises a mixture of at least one emulsifier with a fat or an oil or with both a fat and an oil. Preferably, a hydrophilic emulsifier is included together with a lipophilic emulsifier which acts as a stabiliser. It is also preferred to include both an oil and a fat. Together, the emulsifier(S) with or without stabiliser(s) make up the so-called emulsifying wax, and the wax together with the oil and/or fat make up the so-called emulsifying ointment base which forms the oily dispersed phase of the cream formulations.

Suitable emulgents and emulsion stabilisers include Tween 60, Span 80, cetostearyl alcohol, myristyl alcohol, glyceryl monostearate and sodium lauryl sulfate. The choice of suitable oils or fats for the formulation is based on achieving the desired cosmetic properties, since the solubility of the compound in most oils likely to be used in pharmaceutical emulsion formulations may be very low. Thus the cream should preferably be a non-greasy, non-staining and washable product with suitable consistency to avoid leakage from tubes or other containers. Straight or branched chain, mono- or dibasic alkyl esters such as di-isoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branched chain esters known as Crodamol CAP may be used, the last three being preferred esters. These may be used alone or in combination depending on the properties required. Alternatively, high melting point lipids such as white soft paraffin and/or liquid paraffin or other mineral oils can be used.

Formulations suitable for intranasal administration, where the carrier is a liquid, include, for example, nasal spray, nasal drops, or by aerosol administration by nebuliser, include aqueous or oily solutions of the compound.

Formulations suitable for intranasal administration, where the carrier is a solid, include, for example, those presented as a coarse powder having a particle size, for example, in the range of about 20 to about 500 microns which is administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose.

Formulations suitable for pulmonary administration (e.g., by inhalation or insufflation therapy) include those presented as an aerosol spray from a pressurised pack, with the use of a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichoro-tetrafluoroethane, carbon dioxide, or other suitable gases.

Formulations suitable for ocular administration include eye drops wherein the compound is dissolved or suspended in a suitable carrier, especially an aqueous solvent for the compound.

Formulations suitable for rectal administration may be presented as a suppository with a suitable base comprising, for example, natural or hardened oils, waxes, fats, semi-liquid or liquid polyols, for example, cocoa butter or a salicylate; or as a solution or suspension for treatment by enema.

Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the compound, such carriers as are known in the art to be appropriate.

Formulations suitable for parenteral administration (e.g., by injection), include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions), in which the compound is dissolved, suspended, or otherwise provided (e.g., in a liposome or other microparticulate). Such liquids may additional contain other pharmaceutically acceptable ingredients, such as anti-oxidants, buffers, preservatives, stabilisers, bacteriostats, suspending agents, thickening agents, and solutes which render the formulation isotonic with the blood (or other relevant bodily fluid) of the intended recipient. Examples of excipients include, for example, water, alcohols, polyols, glycerol, vegetable oils, and the like. Examples of suitable isotonic carriers for use in such formulations include Sodium Chloride Injection, Ringers Solution, or Lactated Ringer's Injection. Typically, the concentration of the compound in the liquid is from about 1 ng/mL to about 10 μg/mL, for example from about 10 ng/mL to about 1 μg/mL. The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.

Dosage

It will be appreciated by one of skill in the art that appropriate dosages of the IP compounds, and compositions comprising the IP compounds, can vary from patient to patient. Determining the optimal dosage will generally involve the balancing of the level of therapeutic benefit against any risk or deleterious side effects. The selected dosage level will depend on a variety of factors including, but not limited to, the activity of the particular IP compound, the route of administration, the time of administration, the rate of excretion of the IP compound, the duration of the treatment, other drugs, compounds, and/or materials used in combination, the severity of the condition, and the species, sex, age, weight, condition, general health, and prior medical history of the patient. The amount of IP compound and route of administration will ultimately be at the discretion of the physician, veterinarian, or clinician, although generally the dosage will be selected to achieve local concentrations at the site of action which achieve the desired effect without causing substantial harmful or deleterious side-effects.

Administration can be effected in one dose, continuously or intermittently (e.g., in divided doses at appropriate intervals) throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the formulation used for therapy, the purpose of the therapy, the target cell(s) being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician, veterinarian, or clinician.

In general, a suitable dose of the IP compound is in the range of about 10 μg to about 250 mg (more typically about 100 μg to about 25 mg) per kilogram body weight of the subject per day. Where the compound is a salt, an ester, an amide, a prodrug, or the like, the amount administered is calculated on the basis of the parent compound and so the actual weight to be used is increased proportionately.

EXAMPLES

The following examples are provided solely to illustrate the present invention and are not intended to limit the scope of the invention, as described herein.

Chemical Synthesis

All starting materials, reagents and solvents for reactions were reagent grade and used as purchased. Chromatography solvents were HPLC grade and were used without further purification. Reactions were monitored by thin layer chromatography (TLC) analysis using Merck silica gel 60 F-254 thin layer plates. Flash column chromatography was carried out on Merck silica gel 60 (0.015-0.040 mm) or in disposable Isolute Flash Si and Si II silica gel columns. Preparative TLC was performed on either Macherey-Nagel [809 023] pre-coated TLC plates SIL G-25 UV₂₅₄ or Analtech [2015] pre-coated preparative TLC plates, 2000 μm with UV₂₅₄. LCMS analyses were performed on a Micromass LCT/Water's Alliance 2795 HPLC system with a Discovery 5 μm, C18, 50 mm×4.6 mm i.d. column from Supelco at a temperature of 22° C. using the following solvent systems: Solvent A: Methanol; Solvent B: 0.1% formic acid in water at a flow rate of 1 mL/min. Gradient starting with 10% A/90% B from 0-0.5 minutes then 10% A/90% B to 90% A/10% B from 0.5 minutes to 6.5 minutes and continuing at 90% A/10% B up to 10 minutes. From 10-10.5 minutes the gradient reverted back to 10% A/90% where the concentrations remained until 12 minutes. UV detection was at 254 nm and ionisation was positive or negative ion electrospray. Molecular weight scan range is 50-1000. Samples were supplied as 1 mg/mL in DMSO or methanol with 3 μL injected on a partial loop fill. NMR spectra were recorded in DMSO-d₆ on a Bruker Advance 500 MHz spectrometer.

Synthesis 1 Tert-butyl 2-fluoro-4-hydroxyphenylcarbamate

4-Amino-3-fluorophenol (10.61 g, 83.5 mmol) was added to a molten mixture of Boc₂O (18.29 g, 83.8 mmol) and InCl₃ (188 mg, 0.85 mmol) at 35° C. The black mixture was stirred at 35° C. for 2 hours, during which time it turned into a thick black oil. The mixture was then diluted with EtOAc (200 mL) and H₂O (200 mL) and stirring was continued for 10 minutes. The layers were separated and the organic layer was washed with H₂O (3×200 mL), dried (MgSO₄), filtered and concentrated to dryness. The resulting black oil was redissolved in CH₂Cl₂ (50 mL) and loaded onto a silica gel column. Elution with 5→7% EtOAc in CH₂Cl₂ furnished the title compound as a light yellow, crystalline solid.

Yield: 16.7 g (90%). ¹H-NMR (DMSO-d₆), δ (ppm), J (Hz): 1.42 (s, 9H, C(CH₃)₃), 6.50-6.57 (m, 2H, ArH), 7.11-7.21 (m, 1H, ArH), 8.45 (bs, 1H, OH), 9.63 (s, 1H, NHBoc); ¹³C-NMR (DMSO-d₆), δ (ppm), J (Hz): 28.0, 78.6, 1027 (d. J_(FH)=22.2), 110.8 (d, J_(FH)=2.7), 117.1 (d, J_(FH)=12.6), 127.2, 153.7, 155.5 (d, J_(FH)=11.3), 156.1 (d, J_(FH)=246); ¹⁹F-NMR (DMSO-d₆), δ (ppm): −121.6; LC-MS (3.94 min): m/z calcd. for C₁₁H₁₄FNO₃ [M-C(CH₃)₃]⁺: 172.0; found: 172.0.

Synthesis 2 Tert-butyl 4-(2-amino-3-nitropyridin-4-yloxy)-2-fluorophenylcarbamate (3a)

Dry DMSO (20 mL) was added to NaH (1.029 g of a 60% dispersion in mineral oil, 25.7 mmol) in a round bottom flask under argon. After 5 minutes, solid tert-butyl 2-fluoro-4-hydroxyphenylcarbamate (5.59 g, 24.6 mmol) was added in three portions, giving a dark solution, which, after 15 minutes of stirring at room temperature, was treated with 4-chloro-3-nitropyridin-2-amine (4.23 g, 24.4 mmol) at once. The dark red solution was heated to 110° C. for 1 hour and allowed to cool down to room temperature. EtOAc (150 mL) and H₂O (200 mL) were subsequently added to the solution and the organic layer was isolated. The aqueous layer was extracted with EtOAc (3×100 mL) and the combined organic layers were washed once with saturated NaHCO₃ (150 mL), dried (MgSO₄), filtered, and concentrated to dryness to give a bright yellow solid. This material was used in the next step without further purification.

Yield: 8.68 g (98%). ¹H-NMR (DMSO-d₆), δ (ppm), J (Hz): 1.46 (s, 9H, C(CH₃)₃), 6.08 (d, 1H, ³J_(HH)=5.5, PyrH), 7.01 (m, 1H, ArH), 7.18 (br s, 2H, NH₂), 7.22 (m, 1H, ArH), 7.67 (m, 1H, ArH), 8.04 (d, 1H, ³J_(HH)=5.5, PyrH), 9.03 (s, 1H, NHBoc); ¹⁹F-NMR (DMSO-d₆), δ (ppm): −120.7; LC-MS (4.72 min): m/z calcd. for C₁₆H₁₇FN₄O₆ [M+H⁺]: 365.0; found: 365.0.

Synthesis 3 Tert-butyl 4-(2,3-diaminopyridin-4-yloxy)-2-fluorophenylcarbamate (4a)

Pd/C (1.09 g) was added to a yellow solution of tert-butyl 4-(2-amino-3-nitropyridin-4-yloxy)-2-fluorophenylcarbamate (3a) (6.20 g, 17.0 mmol) in EtOAc/EtOH (90/150 mL) and the black mixture was stirred under a nitrogen atmosphere for 5 hours and filtered over Celite. The dark brown filtrate was concentrated to dryness, redissolved in CH₂Cl₂ (20 mL) and loaded onto a silica gel column. The products were eluted with EtOAc and the fractions containing the title compound were evaporated to dryness. The orange oil was dissolved in CH₂Cl₂ and an equal amount of hexane was added. The solution was concentrated to dryness to give an orange foam.

Yield: 4.30 g (76%). ¹H-NMR (DMSO-d₆), δ (ppm), J (Hz): 1.45 (s, 9H, C(CH₃)₃), 4.47 (s, 2H, NH₂), 5.61 (s, 2H, NH₂), 6.09 (d, 1H, ³J_(HH)=5.5, PyrH), 6.76 (m, 1H, ArH), 6.87 (m, 1H, ArH), 7.28 (d, 1H, ³J_(HH)=5.5, PyrH), 7.47 (m, 1H, ArH), 8.82 (s, 1H, NHBoc); ¹³C-NMR (DMSO-d₆), δ (ppm), J (Hz): 28.0, 79.1, 104.4, 105.9 (d, J_(FH)=23.1), 113.4 (d, J_(FH)=3.1), 120.3, 121.5 (d, J_(FH)=12.2), 126.1, 135.7, 146.2, 150.5, 153.1 (d, J_(FH)=10.1), 153.3, 155.1 (d, J_(FH)=248); ¹⁹F-NMR (DMSO-d₆), δ (ppm): −120.7; LC-MS (2.69 min): m/z calcd. for C₁₆H₂₀FN₄O₃ [M+H⁺]: 335.2; found: 335.3.

Synthesis 4 Ethyl 4-(4-N-(tert-butoxycarbonyl)-amino-3-fluorophenoxy)-2-aminopyridin-3-yl-carbamate (5a)

4-(4-N-(tert-butoxycarbonyl)-amino-3-fluorophenyloxy)-2,3-diaminopyridine (4a) (2.5 g, 7.5 mmol) was dissolved in dry THF (50 mL) under stirring, pyridine (1.2 mL, 15 mmol) was added and the solution was cooled at 0° C. Ethyl chloroformate (0.77 mL, 8.0 mmol) was added at once. After 30 minutes, the reaction mixture was allowed to reach room temperature and stirred for further 24 hours. The solvent was evaporated under vacuum and the residue partitioned between DCM and saturated aqueous Na₂CO₃. The organic layer was washed with H₂O, dried over MgSO₄ and evaporated. The residue was purified by column chromatography (eluent gradient DCM to EtOAc) to afford the title compound as a foam.

Yield: 1.13 g, 37%. ¹H-NMR δ: 1.17 (t, 3H, CH_(3,Et), J=7.1 Hz), 1.45 (s, 9H, tBu), 4.02 (q, 2H, J=7.1, CH_(2,Et)), 5.89 (s, 2H, NH_(2,Py2)), 5.98 (d, 1H, J=5.7, H_(Py)), 6.83 (d, 1H, H_(arom)), 6.96 (d, 1H, H_(arom)), 7.55 (t, 1H, H_(arom)), 7.73 (d, 1H, J=5.7, H_(Py)), 8.29 (br s, 1H, NH_(Py3)), 9.39 (s, 1H, NHBoc).

Synthesis 5 Ethyl 4-(4-amino-3-fluorophenoxy)-2-aminopyridin-3-yl-carbamate (6a)

Ethyl 4-(4-N-(tert-butoxycarbonyl)-amino-3-fluorophenoxy)-2-aminopyridin-3-yl-carbamate (5a) (1.13 g, 2.8 mmol) was dissolved in TEA (8 mL), a few drops of water were added and the reaction mixture was stirred for 2 hours at room temperature. The TEA was evaporated, the residue dissolved in water (20 mL), neutralized with saturated aqueous Na₂CO₃ and extracted with DCM (2×20 mL). The organic layer was dried and evaporated to afford the title compound.

Yield: 730 mg, 86%. ¹H-NMR δ: 1.17 (t, 3H, J=7.1, CH_(3,Et)), 4.05 (q, 2H, J=7.0, CH_(2,Et)), 5.04 (s, 2H, NH_(2,Ph)), 5.71 (s, 2H, NH_(2,Py)), 5.86 (d, 1H, J=5.7, H_(Py)), 6.64 (d, 1H, H_(arom)), 6.73-6.82 (m, 2H, H_(arom)), 7.68 (d, 1H, J=5.7, H_(Py)), 8.23 (br s, 1H, NH_(Py3)). LC-MS: m/z 307 ([M+H]⁺, 100).

Synthesis 6 Ethyl 4-(4-amino-3-fluorophenoxy)-2-aminopyridin-3-yl-methyl-carbamate (7a)

Ethyl 4-(4-amino-3-fluorophenoxy)-2-aminopyridin-3-yl-carbamate (6a) (480 mg, 1.6 mmol) was dissolved in dry THF (8 mL) and cooled at 0° C. Sodium hydride (60% in mineral oil, 80 mg, 2.0 mmol) was added, and the reaction mixture was stirred for 40 minutes at 0° C. Methyl iodide (130 μL, 1.8 mmol) was added at 0° C. The ice bath was removed, and the mixture was stirred at room temperature for 18 hours. The solvent was evaporated and the residue partitioned between DCM and distilled water. The organic layer was dried and evaporated, and the residue washed with diethyl ether to afford the title compound as a brown solid.

Yield: 322 mg, 63%. ¹H-NMR δ: 1.09 (t, 3H, J=7.0, CH_(3,Et)), 3.00 (s, 3H, CH₃N), 3.90-4.10 (m, 2H, CH_(2,Et)), 5.07 (s, 2H, NH_(2,Ph)), 5.87 (d, 1H, Hp), 6.03 (s, 2H, NH_(2,Py)), 6.63 (t, 1H, H_(arom)) 6.77-6.81 (m, 2H, H_(arom)), 7.75 (d, 1H, H_(Py)).

Synthesis 7 7-(4-Amino-3-fluorophenoxy)-1-N-methyl-1H-imidazo[4,5-b]pyridin-2(3H)-one (8a)

Ethyl 4-(4-amino-3-fluorophenoxy)-2-aminopyridin-3-yl-methyl-carbamate (7a) (320 mg, 1.0 mmol) was suspended in a solution of EtONa in EtOH (4 mL), obtained from dissolving sodium (480 mg, 21 mmol) in ethanol (9 mL). The suspension was heated under microwave irradiation for 1 hour (100° C., 100 W). The mixture was cooled at room temperature and the solvent was evaporated. The residue was dissolved in water and was acidified with AcOH to pH 4. The precipitate formed was recovered by filtration, to afford the title compound.

Yield: 188 mg, 67%. ¹H-NMR δ: 3.46 (s, 3H, CH₃N), 5.11 (s, 2H, NH₂), 6.35 (d, 1H, J=5.9, H_(Py)), 6.77-6.82 (m, 2H, H_(arom)), 6.99 (d, 1H, H_(arom)), 7.76 (d, 1H, J=6.0, Hp), 11.54 (s, 1H, NH_(Py)).

Synthesis 8 1-(3-Tert-butyl-1-phenyl-1H-pyrazol-5-yl)-3-(2-fluoro-4-(1-N-methyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-7-yloxy)phenyl)urea (AA-01)

3-Tert-butyl-1-phenyl-1H-pyrazol-5-amine (550 mg, 1.8 mmol) was dissolved in CH₂Cl₂ (25 mL) and an equal volume of saturated NaHCO₃ (aq) was added. The biphasic mixture was stirred and cooled to 0° C. with an ice/water bath. After 10 minutes, 2 equiv. of a 1.9 M solution of phosgene in toluene were added. The mixture was vigorously stirred for 10 minutes, the organic layer was isolated, washed with H₂O, dried (MgSO₄) and concentrated to about 5 mL. This solution was added to a solution of the 7-(4-amino-3-fluorophenoxy)-1-N-methyl-1H-imidazo[4,5-b]pyridin-2(3H)-one (8a) (200 mg, 1.4 mind) in THF. The solution was stirred for 15 hours at room temperature, the solvents were evaporated and the solid residue was washed with Et₂O and CH₂Cl₂ to afford the title compound as a white solid.

Yield: 200 mg, 53%. ¹H NMR, δ: 1.28 (s, 9H, tert-Bu), 3.42 (s, 3H, CH₃), 6.39 (s, 1H, H_(Pyz,4)), 6.49 (d, 1H, J=5.9, H_(Py,5)), 6.99 (dd, 1H, J=1.6, 9.0, H_(arom)), 7.21 (dd, 1H, J=11.9, 2.7, H_(arom)), 7.40-7.45 (m, 1H, H_(arom)), 7.50-7.58 (m, 4H, H_(arom)), 7.81 (d, 1H, J=5.9, H_(Py,6)), 8.10 (t, 1H, J=9.1, H_(arom)), 8.80 (s, 1H, NH_(urea)), 8.93 (s, 1H, NH_(urea)), 11.63 (bs, 1H, NH_(Py2)). LC-MS: m/z 516 ([M]⁺, 100). HRMS (El): m/z calcd for C₂₇H₂₇N₇O₃ ([M+H]⁺): 516.2154. found: 516.2152.

Synthesis 9 1-(3-Tert-butyl-1-p-tolyl-1H-pyrazol-5-yl)-3-(2-fluoro-4-(1-N-methyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-7-yloxy)phenyl)urea (AA-02)

The title compound was prepared from 3-tertbutyl-1-p-tolyl-1H-pyrazol-5-amine (458 mg, 2 mmol) and 7-(4-amino-3-fluorophenoxy)-1-N-methyl-1H-imidazo[4,5-b]pyridin-2(3H)-one (8a) (110 mg, 0.4 mmol) by the same method as described for (AA-01), as an off-white solid.

Yield: 150 mg, 71%. ¹H NMR, δ: 1.27 (s, 9H, tert-Bu), 2.38 (s, 3H, Ph-CH₃), 3.42 (s, 3H, N—CH₃), 6.37 (s, 1H, H_(Pyz,4)), 6.49 (d, 1H, J=5.9, H_(Py,5)), 6.96 (dd, 1H, H_(arom)), 7.20 (d, 1H, H_(arom)), 7.34 (d, 2H, J=8.3, H_(arom)), 7.39 (d, 2H, J=8.4, H_(arom)), 7.81 (d, 1H, J=5.9, H_(Py,6)), 8.11 (t, 1H, H_(arom)), 8.74 (s, 1H, NH_(urea)), 8.92 (s, 1H, NH_(urea)), 11.61 (bs, 1H, NH_(Py2)). LC-MS: m/z 530 ([M+H]⁺, 100).

Synthesis 10 1-(3-Tert-butyl-1-(4-chlorophenyl)-1H-pyrazol-5-yl)-3-(2-fluoro-4-(1-N-methyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-7-yloxy)phenyl)urea (AA-03)

The title compound was prepared from 3-tert-butyl-1-(4-chlorophenyl)-1H-pyrazol-5-amine (375 mg, 1.5 mmol) and 7-(4-amino-3-fluorophenoxy)-1-N-methyl-1H-imidazo[4,5-b]pyridin-2(3H)-one (8a) (150 mg, 0.55 mmol) by the same method as described for (AA-01), as an off-white solid.

Yield: 190 mg, 63%. ¹H NMR, δ: 1.28 (s, 9H, tert-Bu), 3.42 (s, 3H, CH₃), 6.39 (s, 1H, H_(Pyz,4)), 6.49 (d, 1H, J=5.9, H_(Py,5)), 6.96 (dd, 1H, 9.0, H_(arom)), 7.21 (d, 1H, H_(arom)), 7.57 (d, 2H, J=9.0, H_(arom)), 7.60 (d, 2H, J=8.9, H_(arom)), 7.81 (d, 1H, J=5.9, H_(Py,6)), 8.08 (t, 1H, H_(arom)), 8.80 (s, 1H, NH_(urea)), 8.89 (s, 1H, NH_(urea)), 11.63 (bs, 1H, NH_(Py2)). LC-MS: m/z 549 ([M]⁺, 100).

Synthesis 11 3-Tert-butyl-1-(3-fluorophenyl)-1H-pyrazole-5-carboxylic acid

In a round-bottomed flask (dried in an oven) (3-fluorophenyl) boronic acid (224 mg, 1.6 mmol), ethyl-3-t-butyl-pyrazol-5-carboxylate (320 mg, 1.6 mmol), copper acetate (355 mg, 1.9 mmol) and dry pyridine (158 μL, 1.9 mmol) were suspended under vigorous stirring and an argon atmosphere in 10 mL dry DMF. To the reaction mixture, 300 mg of 4 A molecular sieve was added and the suspension stirred for 20 hours at room temperature. The suspension was diluted with 20 mL AcOEt, washed with water (2×20 mL), then with 20 mL conc. NaHCO₃ solution, and 20 mL brine, dried (MgSO₄) and evaporated under vacuum. A thick oil was obtained (520 mg) which was used in the next step without purification.

The oil was dissolved in 10 mL EtOH and 3 mL NaOH solution (2 M) was added under stirring and the reaction mixture was refluxed for 30 minutes. After cooling to room temperature, the reaction mixture was adjusted to pH 4 (with AcOH) and extracted with 20 mL AcOEt. The organic layer was washed with water (2×20 mL), dried and evaporated under vacuum. A solid was obtained (457 mg). The solid was purified on Biotage using cyclohexane:AcOEt 3:1 to afford the title compound as a white solid.

Yield: 166 mg (40%). ¹H NMR (500 MHz, DMSO-d₆): δ 1.30 (s, 9H), 6.95 (s, 1H, H_(pyr)), 8.59 (d, 1H, J=8.6 Hz), 7.25-7.32 (m, 1H, H_(arom)), 7.35 (d, 1H, J=9.9 Hz, H_(arom)), 7.47-7.52 (m, 1H, H_(arom)), 13.22 (s, 1H, H_(acid)). HRMS: (M+H)⁺ calcd for C₁₄H₁₅FN₂O₂, 262.1118, found: 262.1117.

Synthesis 12 1-(3-Tert-butyl-1-(3-fluorophenyl)-1H-pyrazol-5-yl)-3-(2-fluoro-4-(1-methyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-7-yloxy)phenyl)urea (AA-04)

3-Tert-butyl-1-(3-fluorophenyl)-1H-pyrazole-5-carboxylic acid (393 mg, 1.5 mmol) was dissolved in dry DMF (8 mL), and triethylamine (209 μL, 1.5 mmol) was added. The solution was cooled at 0° C., and diphenylphosphoryl azide (323 μL, 1.5 mmol) was added. The reaction mixture was stirred for 30 minutes at 0° C., followed by 1 hour at room temperature. 7-(4-Amino-3-fluorophenoxy)-1-N-thyl-1H-imidazo[4,5-b]pyridin-2(3H)-one (8a) (140 mg, 0.5 mmol) was added, and the reaction mixture heated at 110° C. for 1 hour. The solution was cooled, diluted with AcOEt (100 mL) and extracted with water and brine. The organic layer was dried and evaporated, and the residue taken up in DCM. The remaining solid was recovered by filtration to afford the title compound.

Yield: 25 mg, 9%. ¹H NMR, δ: 1.28 (s, 9H, tert-Bu), 3.41 (s, 3H, CH₃), 6.40 (s, 1H, H_(Pyz,4)), 6.49 (d, 1H, J=6.1, H_(Py,5)), 6.96-7.02 (m, 1H, H_(arom)), 7.19-7.26 (m, 2H, H_(arom)), 7.36-7.44 (m, 2H, H_(arom)), 7.58 (t, 1H, H_(arom)), 7.81 (d, 1H, J=5.9, H_(Py,6)), 8.06 (t, 1H, J=9.1, H_(arom)), 8.83 (s, 1H, NH_(urea)), 8.92 (s, 1H, NH_(urea)), 11.64 (bs, 1H, NH_(Py2)). LC-MS: m/z 533 ([M]⁺, 100).

Synthesis 13 Tert-butyl-4-(2-amino-3-nitropyridin-4-yl-oxy)naphthalen-1-yl-carbamate (3b)

The title compound was prepared from tert-butyl 4-hydroxynaphthalen-1-ylcarbamate (Regan, J. et al, J. Med. Chem., 2002, Vol. 45, No. 14, p. 2994) (3.9 g, 15 mmol) by the same method as described for compound (3a).

Yield: 5.4 g, 90%, upon recrystallization from dichloromethane. ¹H-NMR (DMSO-d₆), δ (ppm), J (Hz): 1.52 (s, 9H, C(CH₃)₃), 5.80 (d, 1H, J=5.7, PyrH), 7.26 (s, 2H, NH₂), 7.38 (d, 1H, J=8.3, ArH, Naph), 7.58-7.69 (m, 3H, ArH, Naph), 7.86-7.89 (m, 1H, ArH(Naph), 7.93 (d, 1H, J=5.5, PyrH), 8.14-8.17 (m, 1H, ArH, Naph), 9.36 (s, 1H, NHBoc).

Synthesis 14 Tert-butyl-4-(2,3-diaminopyridin-4-yl-oxy)naphthalen-1-yl-carbamate (4b)

The title compound was prepared from tert-butyl-4-(2-amino-3-nitropyridin-4-yl-oxy)naphthalen-1-yl-carbamate (3b) (0.50 g, 1.26 mmol) by the same method as described for compound (4a) as a brown solid.

Yield: 0.38 g (82%). ¹H-NMR (DMSO-d₆), δ (ppm), J (Hz): 1.55 (s, 9H, C(CH₃)₃), 4.63 (s, 2H, NH₂), 5.66 (s, 2H, NH₂), 5.92 (d, 1H, J=5.6, PyrH), 7.05 (d, 1H, J=8.3, ArH, Naph), 7.24 (d, 1H, J=5.5, PyrH), 7.54 (d, 1H, J=8.3, ArH, Naph), 7.60-7.65 (m, 2H, ArH, Naph), 8.07-8.12 (m, 2H, ArH, Naph), 9.22 (s, 1H, NHBoc).

Synthesis 15 4-(4-N-Boc-aminonaphthalen-1-yl-oxy)-3-N-aminocarbamoylethyl-2-amino-pyridine (5b)

4-(4-N-Boc-aminonaphthalen-1-yl-oxy)-2,3-diaminopyridine (4b) (500 mg, 1.4 mmol) and the pyridine (222 μL, 2.7 mmol) were dissolved in dry THF (8 mL) under vigorous stirring at 0° C. To this solution the ethylchloroformate (136 mL, 1.5 mmol) was added at once. The reaction mixture was allowed to reach room temperature and was stirred for an additional 10 hours. The solvent was evaporated under vacuum and the residue partitioned between EtOAc and Na₂CO₃ solution. The organic layer was washed (20 mL brine), dried (MgSO₄) and evaporated to provide a solid residue. After purification by LC (Isolute column, Flash Si II, 50 g/170 mL; eluent: EtOAc), the desired compound was obtained.

Yield: 475 mg, 75%. ¹H-NMR δ: 1.14-1.21 (m, 3H, CH₃), 1.50 (s, 9H, tBu), 4.04-4.10 (m, 2H, CH₂), 5.66 (d, 1H, J=5.7, H_(Py)), 5.84 (s, 2H, NH₂), 7.15 (d, 1H, J=8.1, H_(arom)), 7.49-7.60 (m, 3H, H_(arom)), 7.62 (d, 1H, J=5.7, H_(Py)), 7.98-8.04 (m, 1H, H_(arom)), 8.07 (d, 1H, J=8.5, H_(arom)), 8.40 (s, 1H, NH_(carb)) 9.22 (s, 1H, NH_(Boc)). LC-MS: m/z 440 [(M+H)⁺, 100]. HRMS, (El): m/z calcd for C₂₃H₂₇N₄O₅ [(M+H)⁺]: 439.1981; found 439.1979.

Synthesis 16 4-(4-Aminonaphthalen-1-yl-oxy)-3-N-aminocarbamoylethyl-2-amino-pyridine (6b)

4-(4-N-Boc-aminonaphthalen-1-yl-oxy)-3-N-aminocarbamoylethyl-2-aminopyridine (5b) (475 mg, 1.05 mmol) was dissolved in dry TFA (10 mL) under vigorous stirring at 0° C. The solution was allowed to reach room temperature and was stirred for an additional 2 hours. The TFA was evaporated under vacuum and the oily residue partitioned between EtOAc and Na₂CO₃ solution. The organic layer was washed (20 mL brine), dried (MgSO₄) and evaporated to provide a solid residue.

Yield: 346 mg, 97%. ¹H-NMR δ: 1.19-1.26 (m, 3H, CH₃), 4.07-4.13 (m, 2H, CH₂), 5.57 (d, 1H, J=5.7, H_(Py)), 5.77 (s, 4H, 2×NH₂), 6.66 (d, 1H, J=8.1, H_(arom)), 6.97 (d, 1H, J=8:1, H_(arom)), 7.37-7.45 (m, 2H, H_(arom)), 7.55 (d, 1H, J=5.7, H_(Py)), 7.76-7.86 (bs, 1H, H_(arom)), 8.09-8.12 (m, 1H, H_(arom)), 8.36 (s, 1H, NH_(carb)). LC-MS: m/z 339 [(M+H)⁺, 100]. HRMS (El): m/z calcd for C₁₃H₁₉N₄O₃ [(M+H)⁺, 100]: 339.1457; found 339.1459.

Synthesis 17 4-(4-Aminonaphthalen-1-yl-oxy)-3-N-methyl-N-aminocarbamoylethyl-2-amino-pyridine

4-(4-Aminonaphthalen-1-yl-oxy)-3-N-aminocarbamoylethyl-2-amino-pyridine (6b) (350 mg, 1.04 mmol) was dissolved in dry THF (8 mL) under vigorous stirring at 0° C. and argon. To this solution NaH (60% dispersed in mineral oil) (45 mg, 1.14 mmol) was added. After 40 minutes, Mel (66 mL, 0.91 mmol) was added at 0° C. The reaction mixture was allowed to reach room temperature and was stirred for further 10 hours. The solvent was evaporated under vacuum and the residue retaken in 20 mL of EtOAc. The solution was washed with brine (2×20 mL), dried and evaporated to dryness. The residue was triturated with Et₂O and filtered to give the title compound as a solid.

Yield: 238 mg, 65%. ¹H-NMR δ: 1.12-1.29 (m, 3H, CH₃), 3.14 (s, 3H, CH₃), 4.05-4.16 (m, 2H, CH₂), 5.53 (d, 1H, J=5.8, H_(Py)), 5.75 (s, 2H, NH₂), 6.01 (s, 2H, NH₂), 6.66 (d, 1H, J=8.1, H_(arom)), 6.96 (d, 1H, J=8.1, H_(arom)), 7.37-7.43 (m, 2H, H_(arom)), 7.57 (d, 1H, J=5.8, H_(Py)), 7.59-7.64 (m, 1H, H_(arom)), 8.11-8.15 (m, 1H, H_(arom)). LC-MS: m/z 353 ([M+H]⁺, 100). HRMS (El): m/z calcd for C₁₉H₂₁N₄O₃ ([M+H]⁺): 353.1614. found 353.1610.

Synthesis 18 7-(4-Aminonaphthalen-1-yl-oxy)-1-N-methyl-1-H-imidazo[4,5-b]pyridine-2(3H)-one (8b)

230 mg (0.65 mmol) 4-(4-aminonaphthalen-1-yl-oxy)-3-N-methyl-N-aminocarbamoylethyl-2-aminopyridine (7b) were suspended in 5.0 mL solution 1.0 M of EtONa in EtOH. The suspension was submitted to microwave (150 W, 100° C.) for 45 minutes. After cooling, the reaction mixture was evaporated to dryness, retaken in 20 mL H₂O, the pH adjusted to 4.5 (AcOH), precipitating the title compound.

Yield: 167 mg, 84%. ¹H-NMR δ: 3.61 (s, 3H, CH₃), 5.79 (s, 2H, NH₂), 6.10 (d, 1H, J=5.9, F_(Py)), 6.68 (d, 1H, J=8.1, H_(arom)), 7.10 (d, 1H, J=8.1, H_(arom)), 7.43-7.48 (m, 2H, H_(arom)), 7.64 (d, 1H, J-5.8, H_(Py)), 7.74-7.81 (m, 1H, 8.12-8.17 (m, 1H, H_(arom)), 11.57 (s, 1H, NH_(Py)). LC-MS: m/z 307 ([M+H]⁺, 100). HRMS (El): m/z calcd for C₁₇H₂₅N₄O₂ ([M+H]⁺): 307.1195. found 307.1188.

Synthesis 19 1-(3-Tert-butyl-1-phenyl-1H-pyrazol-5-yl)-3-(4-(1-methyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-7-yl-oxy)naphthalen-1-yl)urea (BB-01)

The title compound was prepared from 3-tert-butyl-1-phenyl-1H-pyrazol-5-amine (0.18 mmol) and 7-(4-aminonaphthalen-1-yl-oxy)-1-N-methyl-1H-imidazo[4,5-b]pyridine-2(3H)-one (8b) (50 mg, 0.16 mmol) by the same method as described for (AA-01), as an off-white solid.

Yield: 87 mg, 98%. ¹H NMR, δ 1.29 (s, tert-Bu), 3.53 (s, 3H, CH₃), 6.29 (d, 1H, J=5.9, H_(Py,5)), 6.40 (s, 1H, H_(pyr)), 7.24 (d, 1H, J=8.3, H_(arom)), 7.43 (t, 1H, J=7.0 Hz), 7.54-7.63 (m, 5H), 7.66 (t, 1H, J=8.2, H_(arom)), 7.74 (d, 1H, J=5.9, H_(Py,6)), 7.86 (d, 2H, Jμ8.3, H_(arom)), 8.05-8.10 (m, 1H), 8.77 (s, 1H, 9.07 (s, 1H, NH_(urea)), 11.64 (S, 1H, NH_(Py2)). LC-MS: R_(f)=8.25 min; m/z 548.2 (M⁺, 100). HRMS (El): m/z calcd for C₃₁H₃₀N₇O₃ ([M+H]⁺): 548.2410. found: 548.2404.

Synthesis 20 1-(1-N-p-tolyl-3-tert-butyl-pyrazol-5-yl)-3-(4-(2-oxo-2,3-dihydro-1-N-methyl-1H-imidazo[4,5-b]pyridin-7-yl-oxy)-naphthalen-1-yl)urea (BB-02)

The title compound was prepared from 3-tertbutyl-1-p-tolyl-1H-pyrazol-5-amine (35 mg, 0.15 mmol) and 7-(4-aminonaphthalen-1-yl-oxy)-1-N-methyl-1H-imidazo[4,5-b]-pyridin-2(3H)-one (8b) (40 mg, 0.13 mmol) by the same method as described for (AA-01), as an off-white solid.

Yield: 52 mg, 71%. ¹H NMR, δ: 1.28 (s, 9H, tert-Bu), 2.40 (s, 3H, CH₃), 3.53 (s, 3H, CH₃), 6.29 (d, 1H, J=5.9, H_(Py,5)), 6.39 (s, 1H, H_(pyr)), 7.24 (d, 1H, J=8.4, H_(arom)), 7.36 (d, 2H, J=8.2, H_(arom)), 7.45 (d, 2H, J=8.2, H_(arom)), 7.60 (t, 1H, J=7.5, H_(arom)), 7.67 (t, 1H, J=7.6, H_(arom)), 7.74 (d, 1H, J=5.9, H_(Py,6)), 7.87 (d, 1H, J=8.3, H_(arom)), 8.07 (d, 2H, J=8.3, H_(arom)), 8.71 (s, 1H, NH_(urea)) 9.06 (s, 1H, NH), 11.65 (s, 1H, NH_(Py3)). LC-MS: R_(t)=5.23 min; m/z 562.2 ([M+H]⁺, 100). HRMS (El): m/z calcd for C₃₂H₃₂N₇O₃ ([M+H]⁺): 562.2561. found: 562.2566.

(Reference) Synthesis 21 Tert-butyl 2-fluoro-4-(2-(methylamino)-3-nitropyridin-4-yloxy)phenylcarbamate

Tert-butyl 2-fluoro-4-hydroxyphenylcarbamate (3.25 g, 14.4 mmol) was dissolved in DMSO (25 mL) and the solution was stirred under an argon atmosphere for 20 minutes. Sodium hydride (60% in mineral oil, 580 mg, 14.4 mmol) was added portionwise and the dark solution was stirred at room temperature for 1 hour. 4-chloro-N-methyl-3-nitropyridin-2-amine (2.7 g, 14.4 mmol) dissolved in DMSO (5 mL) was added at once and the red solution was stirred at 50° C. for 2 hours. The solution was cooled, poured onto crushed ice (200 g) and extracted with ethyl acetate (3×100 mL). The organic phases were washed with brine, dried and evaporated to give the title compound as a yellow solid.

Yield: 5.3 g, 90%. ¹H NMR (500 MHz, DMSO-d₆) δ: 1.47 (s, 9H, tert-Bu) 2.93 (d, J=4.5 Hz, CH₃N), 6.08 (d, J=57 Hz, 1H, H_(py)), 7.00 (m, 1H, H_(arom)), 7.22 (m, 1H, H_(arom)), 7.55 (q, J=4.5 Hz, 1H, NHCH₃), 7.66 (m, 1H, H_(arom)), 8.14 (d, J=5.7 Hz, 1H, 1H, H_(py)), 9.04 (bs, 1H, NH). LC-MS: m/z 378.3 ([M+H]⁺, 100).

(Reference) Synthesis 22 Tert-butyl 4-(3-amino-2-(methylamino)pyridin-4-yloxy)-2-fluorophenylcarbamate

Tert-butyl 2-fluoro-4-(2-(methylamino)-3-nitropyridin-4-yloxy)phenylcarbamate (5.3 mg, 14 mmol) was dissolved in absolute ethanol (600 mL) and hydrogenated on a 10% Pd/C cartridge through an H-Cube apparatus, to give the title compound as a yellow solid.

Yield: 4.88 g (quantitative yield). ¹H NMR (500 MHz, DMSO-d₆) δ: 1.44 (s, 9H, tert-Bu), 2.85 (d, 3H), J=4.5 Hz, CH₃N), 4.51 (bs, 2H, NH₂), 5.94 (m, 1H, CH₃NH), 6.10 (d, 1H, J=5.6 Hz, H_(py)), 6.72 (m, 1H, H_(arom)), 6.85 (m, 1H, H_(arom)), 7.37 (d, 1H, J=5.6 Hz, H_(p)), 7.44 (m, 1H, H_(arom)), 8.87 (bs, 1H, NH). LC-MS: m/z 348.4 ([M+H]⁺, 100).

Reference Synthesis 23 Tert-butyl 2-fluoro-4-(3-methyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-7-yloxy)phenylcarbamate

To an ice-cooled solution of tert-butyl 4-(3-amino-2-(methylamino)pyridin-4-yloxy)-2-fluorophenylcarbamate (4.9 g, 14 mmol) in THF (150 mL) and pyridine (10 mL) under an argon atmosphere, a solution of triphosgene (4.45 g, 15 mmol) in THF (75 mL) was added over 2 hours via a dropping funnel. The solution was stirred for 2 hours at 0° C., followed by 4 hours at room temperature, and then refluxed overnight. After cooling, the solution was filtered, evaporated and chromatographed on a Biotage apparatus (25+M column, eluent DCM/EtOAc 1/1) to give the title compound as a white solid.

Yield: 2.05 g, 40%. ¹H NMR (500 MHz, DMSO-d₆) δ: 1.47 (s, 9H, tert-Bu), 3.32 (s, 3H, CH₃N), 6.53 (d, 1H, J=5.9 Hz, H_(py)), 6.94 (m, 1H, H_(arom)), 7.15 (m, 1H, H_(arom)), 7.60 (m, 1H, H_(arom)), 7.89 (d, 1H, J=5.9 Hz, H_(py)), 8.97 (s, 1H, NH), 11.46 (s, 1H, NH). LC-MS: m/z 375.1 ([M+H]⁺, 100).

(Reference) Synthesis 24 7-(4-Amino-3-fluorophenoxy)-3-methyl-1H-imidazo[4,5-b]pyridin-2(3H)-one

A solution of tert-butyl 2-fluoro-4-(3-methyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-7-yloxy)phenylcarbamate (2.18 g, 5.8 mmol) in 1 M TBAF in THF (41 mL) was refluxed overnight under an argon atmosphere. The solution was cooled and evaporated and water (20 mL) was added, upon which a precipitate formed. The precipitate was filtered, washed with water, and dried to give the title compound as an off-white solid.

Yield: 1.37 g, 86%. ¹H NMR (500 MHz, DMSO-d₆): δ 3.29 (s, 3H, CH₃N), 5.17 (s, 2H, NH₂), 6.35 (d, 1H, J=5.94 Hz, H_(py)). 6.74-6.83 (m, 2H, H_(arom)), 6.99 (m, 1H, H_(arom)), 7.81 (d, 1H, J=5.94 Hz, H_(py)), 11.44 (s, 1H, NH). LC-MS: m/z 275.1 ([M+H]⁺, 100).

(Reference) Synthesis 25 7-(4-amino-3-fluorophenoxy)-1,3-dimethyl-1H-imidazo[4,5-b]pyridin-2(3H)-one

To a solution of 7-(4-amino-3-fluorophenoxy)-3-methyl-1H-imidazo[4,5-b]pyridin-2(3H)-one (100 mg, 0.365 mmol) in THF (4 mL) at 0° C. under argon, NaH (16.77 mg, 0.419 mmol) was added in one portion. The resulting solution was stirred for 20 minutes and iodomethane (0.025 mL, 0.401 mmol) was added. After 1 hour, water was added, and the solution evaporated and extracted with DCM (3×20 mL) to give the title compound.

Yield: 80 mg, 0.278 mmol, 76%. ¹H NMR (CDCl₃), δ: 3.51 (s, 3H, Me), 3.66 (s, 3H, Me), 6.40 (d, 1H, J=6.0, H_(arom,Py)) 6.74 (ddd, 1H, J=8.6, 2.5, 1.0, H_(arom,FPh)), 6.83 (m, 2H, H_(arom,FPh)) 7.87 (d, 1H, J=6.0, H_(arom,Py)). LC-MS: Rf=2.60 min; m/z 289.1 ([M+H]+, 90).

(Reference) Synthesis 26 1-(1-N-p-tolyl-3-tert-butyl-pyrazol-5-yl)-3-(4-(2-oxo-2,3-dihydro-1-N-methyl-1H-imidazo[4,5-b]pyridin-7-yl-oxy)phenyl)urea (XX-02)

The title compound was prepared using 3-tert-butyl-1-p-tolyl-1H-pyrazol-5-amine (43.7 mg, 0.19 mmol) and 7-(4-aminophenyloxy)-1-N-methyl-1H-imidazo[4,5-b]pyridin-2(3H)-one (Niculescu-Duvaz, D. et al, J. Med. Chem., 2009, Vol. 52, No. 8, p. 2255) (40 mg, 0.16 mmol) by the same method as described for (AA-01), as a white solid.

Yield: 62 mg, 76%. ¹H NMR, δ: 1.27 (s, 9H, tert-Bu), 2.37 (s, 3H, CH₃), 3.44 (s, 3H, CH₃), 6.35 (d, 1H, J=5.9, H_(Py,5)), 6.39 (s, 1H, H_(pyr)), 7.11 (d, 2H, J=9.0, H_(arom)), 7.33 (d, 2H, J=8.3, H_(arom)), 7.39 (d, 2H, J=8.3, H_(arom)), 7.47 (d, 2H, J=9.0, H_(arom)), 7.78 (d, 1H, J=5.9, H_(Py,6)), 8.32 (s, 1H, NH_(urea)), 9.08 (s, 1H, NH_(urea)), 11.59 (s, 1H, NH_(Py3)). LC-MS: R_(f)=5.14 min; m/z 511 ([M+H]⁺, 100). HRMS (El): m/z calcd for C₂₃H₃₀N₇O₃ ([M+H]⁺): 512.2405. found: 512.2405.

(Reference) Synthesis 27 1-(3-Tert-butyl-1-phenyl-1H-pyrazol-5-yl)-3-(4-(1,3-dimethyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-7-yloxy)-2-fluorophenyl)urea (XX-03)

The title compound was prepared from 3-tert-butyl-1-phenyl-1H-pyrazol-5-amine (130 mg, 0.60 mmol) and 7-(4-amino-3-fluorophenoxy)-1,3-dimethyl-1H-imidazo[4,5-b]pyridin-2(3H)-one (100 mg, 0.35 mmol) by the same method as described for (AA-01), as a yellow powder.

Yield: 15 mg, 8%. ¹H NMR (CDCl₃), δ: 1.39 (s, 9H, tert-Bu), 3.48 (s, 3H, Me), 3.59 (s, 3H, Me), 6.47 (d, 1H, J=5.9, H_(arom,Py)), 6.49 (s, 1H, H_(Pyz,4)), 6.85 (dd, 1H, J=11.1, 2.5, H_(arom,FPh)), 6.89 (d, 1H, J=9.0, H_(arom,FPh)), 7.31 (t, 1H, J=7.7, H_(arom,Ph)), 7.42 (t, 2H, J=7.7, H_(arom,Ph)), 7.50 (d, 2H, J=7.9, H_(arom,Ph)) 7.92 (d, 1H, J=5.9, H_(arom,Py)), 8.15 (t, 1H, J=8.9, H_(arom,FPh)) LC-MS: Rf=2.78 min; m/z 530 ([M+H]+, 90). HRMS (El): m/z calcd for C₂₈H₂₉FN₇O₃ ([M+H]+): 530.2310. found: 530.2326.

Reference Synthesis 28 1-(5-Tert-butyl-2-phenyl-2H-pyrazol-3-yl)-3-[4-(2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-7-yloxy)-phenyl]-urea (XX-01)

The title compound was obtained using known methods, as shown, for example, in Synthesis 61 in Niculescu-Duvaz et al., 2006.

(Reference) Synthesis 29 1-[5-tert-Butyl-2-(4-fluoro-phenyl)-2H-pyrazol-3-yl]-3-[4-(2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-7-yloxy)-phenyl]-urea (XX-04)

The title compound was obtained using known methods, as shown, for example, in Synthesis 79 in Niculescu-Duvaz et al., 2006.

Biological Methods Biological Methods—Assay A—DELFIA Kinase Assay

Compounds were assessed by a kinase assay performed according to the following protocol.

The following reagents were prepared:

DELFIA Kinase Buffer (DKB):

TABLE 2 Volume Volume per Stock per mL 10 ml plate Reagent Concentration (μL) (μL) 20 mM MOPS pH 7.2 0.2M 100 1000 0.5M EGTA pH 8.0 0.5M 10 100 10 mM MgCl₂   1M 10 100 0.1% β-mercaptoethanol — 1 10 25 mM β-glycerophosphate 0.5M 50 500 Water 100% 829 8290

MOPS=3-[N-Morpholino] propanesulfonic acid (Sigma M3183).

EGTA=Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (Sigma E3889).

DKB1 (DKB with B-RAF and MEK protein):

Combine 4950 μL of DKB and 50 μL of 2.5 mg/mL GST-MEK stock (t) give 1 mg of MEK per 40 μL). Then add 22.5 μL of B-RAF to give ˜0.2 μL of B-RAF per 40 μL.

DKB2 (DKB with MEK protein):

Combine 4950 μL of DKB and 50 μL of 2.5 mg/mL GST-MEK stock (to give 1 mg of MEK per 40 μL). Use 500 μL of this for the blow out (BO) and the empty vector (EV) control.

ATP:

100 mM stock, dilute to 500 μM to give 100 μM final concentration in assay.

Inhibitors (Test Compounds):

100 mM stock, dilute to 10, 3, 1, 0.3, 0.1, 0.03, 0.01, 0.003, 0.001, 0.0003, and 0.0001 mM in DMSO in drug plate, resulting in concentration of 100, 30, 10, 3, 1, 0.3, 0.1, 0.03, 0.01, 0.003, and 0.001 μM in the assay.

Primary Antibody:

Phospho-MEK1/2 CST #9121S diluted 1:1000 in DELFIA assay buffer (AB). Pre-incubate antibody in the AB for 30 minutes at room temperature prior to use.

Secondary Antibody:

Anti-rabbit-Eur labelled secondary Perkin Elmer #AD0105 diluted 1:1000 in DELFIA assay buffer (AB). Pre-incubate antibody in the AB for 30 minutes at room temperature prior to use. (Primary and secondary antibodies were incubated together.)

Tween:

0.1% Tween 20 in water.

Assay Buffer:

DELFIA assay buffer Perkin Elmer #4002-0010.

Enhancement Solution:

DELFIA enhancement solution Perkin Elmer #4001-0010.

Assay Plates:

96 well glutathione-coated black plate Perbio #15340.

Procedure:

1. Preblock wells with 5% milk in TBS for 1 hour.

2. Wash wells 3× with 200 μL TBS.

3. Plate out 40 μL of DKB1 for all inhibitors (test compounds), DMSO control, and optionally other control compounds.

4. Plate out 40 μL of DKB2 for BO and EV wells.

5. Add inhibitors (test compounds) at 0.5 μL per well according to desired plate layout.

6. Add 0.5 μL DMSO to vehicle control wells.

7. Add 2 μL of B-RAF to BO and EV wells.

8. Pre-incubate with inhibitors (test compounds) for 10 minutes at room temperature with shaking.

9. Add 10 μL of 500 μM ATP stock, in DKB, to give 100 μM assay concentration.

10. Seal plates with TopSeal and incubate at room temperature with shaking for 45 minutes.

11. Wash plates 3× with 200 μL 0.1% Tween20/Water to terminate reaction.

12. Add 50 μL per well of antibody mix and incubate for 1 hour at room temperature with shaking.

13. Wash plates 3× with 200 μL 0.1% Tween20/Water.

14. Add 100 μL DELFIA enhancement solution per well, cover in foil, and incubate at room temperature for 30 minutes with shaking.

15. Read on Victor using Europium protocol.

Values for the blank (Empty Vector) are subtracted from all values. The DMSO controls are set as 100% activity and assay points (the response) are calculated as a percentage of the DMSO control. Data are plotted using Graphpad Prism software and a non-linear regression line is calculated using a variable slope sigmoidal dose-response equation: Y=Bottom+[Top−Bottom]/[1+10^(Log EC50−X)*HillSlope)] where X is the logarithm of concentration and Y is the response. The IC50 generated by this procedure is the concentration of the drug that produces a percentage control fluorescence value midway between the saturation, and zero-effect plateaus. Three independent assays are usually performed and the mean IC50 is reported. Biological Methods—Assay B—Cell Based Phosho-ERK Assay

Compounds were assessed using a cell-based assay which was performed according to the following protocol.

Day 0:

Plate out 16,000 mutant BRAF WM266.4 cells/well in 99 μL medium in a 96-well plate.

Day 1:

1. Add 1 μL inhibitor (test compound) to the cells (total 1 μL solution).

2. Incubate the cells with test compound for 6 hours at 37° C.

3. Aspirate off the solution from all of the wells.

4. Fixate the cells with 100 μL 4% formaldehyde/0.25% Triton X-100 PBS per well.

5. Incubate the plate for 1 hour at 4° C.

6. Aspirate off the fixing solution and add 300 μL TBS per well.

7. Leave the plate overnight at 4° C.

Day 2:

1. Wash the plate 2× with 200 μL PBS per well.

2. Block with 100 μL 5% dried milk in TBS.

3. Incubate the plate for 20 minutes at 37° C.

4. Wash the plate 2× with 0.1% tween/H₂O.

5. Add 50 μL of 3 μg/mL primary antibody pERK (Sigma M8159), diluted in 5% milk powder/TBS, to each well.

6. Incubate the plate for 2 hours at 37° C.

7. Wash the plate 3× with 0.1% tween/H₂O.

8. Add 50 μL of 0.45 μg/mL secondary Europium-labelled anti-mouse antibody (Perkin Elmer) to each well.

9. Incubate the plate for 1 hour at 37° C.

10. Wash the plate 3× with 0.1% tween/H₂O.

11. Add 100 μL enhancement solution (Perkin Elmer) to each well.

12. Leave the plate for approximately 10 minutes at room temperature before gently shaking the plate.

13. Read Europium Time Resolved Fluorescence in Victor2.

14. Wash the plate 2× with 0.1% tween/H₂O.

15. Measure the protein concentration with BCA (Sigma) by adding 200 μL of solution per well.

16. Incubate the plate for 30 minutes at 37° C.

17. Read absorbance levels at 570 nm in a plate reader.

Note that Europium counts are normalised for protein levels by dividing counts by absorbance.

Values for the blank (no cells) are subtracted from all values. The DMSO controls are set as 100% activity and assay points (the response) are calculated as a percentage of the DMSO control. Data are plotted using Graphpad Prism software and a non-linear regression line is calculated using a variable slope sigmoidal dose-response equation: Y=Bottom+[Top−Bottom]/[1+10^(Log EC50−X)*HillSlope)] where X is the logarithm of concentration and Y is the response). The IC50 generated by this procedure is the concentration of the drug that produces a percentage control fluorescence value midway between the saturation, and zero-effect plateaus. Three independent assays are usually performed and the mean IC50 is reported. Biological Methods—Assay C—SRB Cell Proliferation Assay (SRB GI₅₀)

Cell lines (e.g., WM266.4 and A375M melanoma cell lines; SW620 colorectal carcinoma cell line) are routinely cultured in DMEM or RPMI1640 supplemented with 10% foetal bovine serum, at 37° C., in 10% CO₂ water saturated atmosphere. Cultures are maintained in exponential growth phase by sub-culturing before having become confluent (3-5 day intervals). Single cell suspensions are prepared by harvesting an 80 cm² tissue culture flask with 5 mL commercial trypsin EDTA. Alter 5 minutes, the detached cells are mixed with 5 fully complemented culture medium and centrifugally pelleted (1000 rpm for 7 minutes). After aspirating the supernatant, the cell pellet is re-suspended in 10 mL fresh medium and the cells fully disaggregated by drawing the whole volume up/down 5 times through a 19-gauge needle. The concentration of the cells is determined using a haemocytometer (1/10 dilution). A suitable volume to give at least a 2-fold excess for the number of tests being conducted, typically 100-200 mL, is prepared by diluting the cell suspension to 10,000-40,000/mL, and 100 μL/well dispensed into 96 well plates using a programmable 8-channel peristaltic pump, giving 1000-4000 cells/well, leaving column 12 blank. The plates are returned to the incubator for 24 hours to allow the cells to re-attach.

The compounds being tested are prepared at 10 mM in DMSO. Aliquots (24 μL) are diluted into 1.2 mL culture medium giving 200 μM, and 10 serial dilutions of 3× performed by transferring 80 μL to 160 μL. Aliquots (100 μL) of each dilution are added to the wells, using an 8-channel pipettor, thus performing a final further 2× dilution, and giving doses ranging from 100 μM to 0.005 μM. Column 11 receives plain culture medium only. Each compound is tested in quadruplicate, each replicate being the average of four wells.

After a further 5 days growth, the plates are emptied, and the cells are fixed in 10% trichloroacteic acid for 30 minutes at 4° C. After thorough rinsing in running tap water, the plates are dried, and stained by adding 50 μL of a solution of 0.1% sulphorhodamine-B in 1% acetic acid, for 10 minutes at room temperature. The stain is poured out and the plates thoroughly rinsed under a stream of 1% acetic acid, thus removing unbound stain, and dried. The bound stain is taken into solution by addition of 100 μL Tris buffer pH 8, followed by 10 minutes on a plate-shaker (approximately 500 rpm). The absorbance at 540 nm in each well (being proportional to the number of cells present) is determined using a plate reader.

After averaging the blank values in column 12 this was subtracted from all values, and results expressed as a percentage of the untreated value (column 11). The 10 values so derived (in quadruplicate) are plotted against the logarithm of the drug concentration, and analysed by non-linear regression to a four parameter logistic equation, setting constraints if suggested by inspection. The GI₅₀ generated by this procedure is the concentration of the drug that produces a percentage control A₅₄₀ midway between the saturation, and zero-effect plateaus.

Biological Methods—Xenograft Studies

SW620 human colorectal carcinoma cells (mutant RAS, 7×10⁷) or A375M human melanoma celss (mutant BRAF, 10⁷) were inoculated sub-cutaneuously in suspension (0.2 mL) into the right flank of female Crl:CD1-Foxn1nu athymic mice. Groups were assigned to treatment following stratified allocation of tumour volumes. Treatment with test compound began between days 11-14 post-cell administration. For gavage, a suspension (DMSO:water, 1:19, v/v at 10 mL/kg) was administered. Control animals received a similar dosage of vehicle (DMSO: water, 1:19, v/v). Treatment with test compound was continued once daily for 24 doses.

Biological Data—Assay Data

Data for several compounds of the present invention, as well as several comparison compounds are summarised in the following table. Lower IC₅₀/GI₅₀ values indicate higher potency.

TABLE 3 Assay A Assay B Assay C Assay C Assay C BRAF pERK SRB SRB SRB — WM266.4 WM266.4 A375M SW620 Code IC₅₀ (μM) IC₅₀ (μM) GI₅₀ (μM) GI₅₀ (μM) GI₅₀ (μM) AA-01 0.27 0.26 0.052 0.138 0.167 AA-02 0.197 0.020 0.057 AA-03 0.302 0.060 0.121 BB-01 0.25 0.019 0.008 0.052 1.6 BB-02 2.28 0.026 0.016 0.101 1.685 XX-01 0.094 0.62 0.39 1.13 0.84 XX-02 0.20 0.09 0.066 0.25 0.45 XX-03 1.09 0.12 0.033 0.247 6.83 XX-04 0.24 0.20 0.020 0.307 2.72

In the in vitro BRAF enzyme assay (Assay A), the compounds of the present invention (˜0.2-2.3 μM) and the comparison compounds (˜0.1-1.1 μM) had similar BRAF IC₅₀ values.

In the in vitro pERK cell-based assay (Assay B), the compounds of the present invention (˜0.02-0.26 μM) and the comparison compounds (˜0.1-0.6 μM) had similar pERK IC₅₀ values.

In the in vitro SRB cell-based Assay (Assay C), for the mutant BRAF cell lines WM266.4 and A375M, the compounds of the present invention (WM266.4: ˜0.01-0.12 μM; A375M: ˜0.05-0.14 μM) and the comparison compounds (WM266.4: ˜0.02-0.4 μM; A375M: ˜0.25-1.1 μM) had similar G150 values. Notably, BR-01 was the most potent (with the lowest GI₅₀: WM266.4, 0.008 μM; A375M, 0.052 μM), and XX-01 was the least potent (with the highest GI₅₀: WM266.4, 0.39 μM; A375M, 1.13 μM).

In the in vitro SRB cell-based Assay (Assay C), for the mutant RAS cell line SW620, the compounds of the present invention (SW620: ˜0.17-1.6 μM) and the comparison compounds (SW620: ˜0.45-7 μM) had similar GI50 values.

However, note that, based on the in vitro mutant RAS data, it would be expected that XX-01 and XX-02 would have better therapeutic efficacy than BB-02, in the in vivo mutant RAS SW620 xenograft study.

Similarly, based on the in vitro mutant RAS data, it would be expected that XX-02 would have a similar therapeutic efficacy as AA-01, in the in vivo mutant RAS SW620 xenograft study.

Based on the in vitro mutant RAS data, it would not be expected that the claimed compounds (especially AA-01 and BB-02) are substantially more effective than the comparison compounds (XX-01, XX-02, XX-03, XX-04), in the in vivo mutant RAS SW620 xenograft study.

Biological Data—BRAF/RAS/Wild Type Selectivity Data

Data for several compounds of the present invention, as well as several comparison compounds are illustrated in FIGS. 4 to 10, as discussed below. The data illustrate the selectivity, or lack of selectivity, for mutant BRAF or mutant RAS cell lines.

FIG. 4 is a bar graph showing the ratio of the GI₅₀ for each of a range of cell lines to the GI₅₀ for the WM266.4 cell line for compound AA-01. The range of cell lines are (a) a panel of mutant BRAF (mutBRAF) cell lines: WM266.4, A375M, UACC62; (b) a panel of mutant RAS (mutRAS) cell lines: SW620, HCT116, and WM1361; and (c) a panel of wild type BRAF and RAS (wtBRAFT/RAS) cell lines: SKMEL23, KM12, and BT474.

FIG. 5 is a bar graph showing the ratio of the GI₅₀ for each of a range of cell lines to the GI₅₀ for the WM266.4 cell line for compound BB-01. The ranges of cell lines are as for FIG. 4.

FIG. 6 is a bar graph showing the ratio of the GI₅₀ for each of a range of cell lines to the GI₅₀ for the WM266.4 cell line for compound BB-02. The ranges of cell lines are as for FIG. 4.

FIG. 7 is a bar graph showing the ratio of the GI₅₀ for each of a range of cell lines to the GI₅₀ for the WM266.4 cell line for comparison compound XX-01. The ranges of cell lines are as for FIG. 4.

FIG. 8 is a bar graph showing the ratio of the GI₅₀ for each of a range of cell lines to the GI₅₀ for the WM266.4 cell line for comparison compound XX-02. The ranges of cell lines are as for FIG. 4.

FIG. 9 is a bar graph showing the ratio of the GI₅₀ for each of a range of cell lines to the GI₅₀ for the WM266.4 cell line for comparison compound XX-03. The ranges of cell lines are as for FIG. 4.

FIG. 10 is a bar graph showing the ratio of the GI₅₀ for each of a range of cell lines to the GI₅₀ for the WM266.4 cell line for comparison compound XX-04. The ranges of cell lines are as for FIG. 4.

Based on these in vitro data, it would not be expected that the claimed compounds (especially AA-01 and BB-02) are substantially more effective than the comparison compounds (XX-01, XX-02, XX-03, XX-04), in the in vivo mutant RAS SW620 xenograft study.

Biological Data—Xenograft Data

Xenograft data for several compounds of the present invention, as well as several comparison compounds are illustrated in FIGS. 11-19.

FIG. 11 is a graph of relative tumour volume as a function of time (days) for mouse xenografts of the mutant RAS cell line SW620, for treatment with compound AA-01 and for controls.

The data demonstrate that compound AA-01 substantially reduced tumour volume over the timescale of the study (e.g., a factor of about 3), as compared to the control, for this mutant RAS colorectal carcinoma cell line.

FIG. 12 is a graph of relative tumour volume as a function of time (days) for mouse xenografts of the mutant RAS cell line SW620, for treatment with compound BB-02 and for controls.

The data demonstrate that compound BB-02 substantially reduced tumour volume over the timescale of the study (e.g., a factor of about 1.5), as compared to the control, for this mutant RAS colorectal carcinoma cell line.

FIG. 13 is a graph of relative tumour volume as a function of time (days) for mouse xenografts of the mutant BRAF cell line A375, for treatment with compound AA-01 and for controls.

The data demonstrate that compound AA-01 substantially reduced tumour volume over the timescale of the study (e.g., a factor of about 2.5), as compared to the control, for this melanoma cell line.

FIG. 14 is a graph of relative tumour volume as a function of time (days) for mouse xenografts of the mutant BRAF cell line A375, for treatment with compound BB-02 and for controls.

The data demonstrate that compound BB-02 substantially reduced tumour volume over the timescale of the study (e.g., a factor of about 1.5), as compared to the control, for this mutant BRAF melanoma cell line.

FIG. 15 is a graph of relative tumour volume as a function of time (days) for mouse xenografts of the mutant RAS cell line SW620, for treatment with comparison compound XX-01 and for controls.

The data demonstrate that comparison compound XX-01 had relatively little effect, as compared to the control, for this mutant RAS colorectal carcinoma cell line.

FIG. 16 is a graph of relative tumour volume as a function of time (days) for mouse xenografts of the mutant RAS cell line SW620, for treatment with comparison compound XX-02 and for controls.

The data demonstrate that comparison compound XX-02 had relatively little effect, as compared to the control, for this mutant RAS colorectal carcinoma cell line.

FIG. 17 is a graph of relative tumour volume as a function of time (days) for mouse xenografts of the mutant RAS cell line SW620, for treatment with comparison compound XX-03 and for controls.

The data demonstrate that comparison compound XX-03 had relatively little effect, as compared to the control, for this mutant RAS colorectal carcinoma cell line.

FIG. 18 is a graph of relative tumour volume as a function of time (days) for mouse xenografts of the mutant RAS cell line SW620, for treatment with comparison compound XX-04 and for controls.

The data demonstrate that comparison compound XX-04 had relatively little effect, as compared to the control, for this mutant RAS colorectal carcinoma cell line.

FIG. 19 is a graph of relative tumour volume as a function of time (days) for mouse xenografts of the mutant BRAF cell line A375, for treatment with comparison compound XX-02 and for controls.

The data demonstrate that comparison compound XX-02 is at least to some degree effective, as compared to the control, for this mutant BRAF melanoma cell line.

The ratios of tumour volume (for treatment) to tumour volume (for control) (T/C) for mutant RAS SW620 xenografts are shown in the following table. A lower T/C ratio indicates a higher efficacy. Whereas the comparison compounds all have T/C ratios which indicate little or no efficacy, the claimed compounds AA-01 and BB-02 both have T/C ratios which demonstrate statistically significant efficacy.

TABLE 4 Ratio of: Tumour Volume (Treatment)/ Tumour Volume (Control) Code (mutant RAS SW620 xenograft) AA-01 0.34 BB-02 0.66 XX-01 >1 XX-02 0.95 XX-03 0.97 XX-04 0.87

The data for the claimed compounds demonstrate that, not only are the claimed compounds effective as BRAF inhibitors and against mutant BRAF tumours (for example, xenografts of the mutant BRAF melanoma cell line A375M; FIGS. 13 and 14), but, surprisingly and unexpectedly, the claimed compounds are also effective against mutant RAS tumours (for example, xenografts of the mutant RAS colorectal carcinoma cell line SW620; FIGS. 11 and 12). The surprising and unexpected activity of the claimed compounds against mutant RAS tumours could not be predicted from their known or expected BRAF inhibitory activity.

The data for the comparison compounds demonstrate that, although the comparison compounds are to some degree effective against mutant BRAF tumours (for example, xenografts of the mutant BRAF melanoma cell line A375M; FIG. 19), they have relatively little effect against mutant RAS tumours (for example, xenografts of the mutant RAS colorectal carcinoma cell line SW620; FIGS. 15, 16, 17, and 18).

The foregoing has described the principles, preferred embodiments, and modes of operation of the present invention. However, the invention should not be construed as limited to the particular embodiments discussed. Instead, the above-described embodiments should be regarded as illustrative rather than restrictive, and it should be appreciated that variations may be made in those embodiments by workers skilled in the art without departing from the scope of the present invention.

REFERENCES

A number of patents and publications are cited herein in order to more fully describe and disclose the invention and the state of the art to which the invention pertains. Full citations for these references are provided below. Each of these references is incorporated herein by reference in its entirety into the present disclosure, to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference.

-   Bos, 1989, “ras oncogenes in human cancer: a review”, Cancer Res.,     Vol. 49, pp. 4682-4689. -   Downward, 2003, “Targeting RAS signalling pathways in cancer     therapy”, Nat. Rev. Cancer, Vol. 3, pp. 11-22. -   Garnett et al., 2004, “Guilty as charged: B-RAF is a human     oncogene”, Cancer Cell, Vol. 6, pp. 313-319. -   Gray-Schopfer et al., 2007, “Melanoma biology and new targeted     therapy”, Nature, Vol. 445, pp. 851-857. -   Niculescu-Duvaz et al., 2006, “Imidazo[4,5-b]pyridine-2-one and     oxazolo[4,5-b]pyridine-2-one compounds and analoges thereof as     therapeutic compounds”, international patent application publication     number WO 2006/043090 A1 published 27 Apr. 2006. -   Niculescu-Duvaz et al., 2007, “Imidazo[4,5-b]pyridine-2-one and     oxazolo[4,5-b]pyridine-2-one compounds and analoges thereof as     cancer therapeutic compounds”, international patent application     publication number WO 2007/125330 A1 published 8 Nov. 2007. -   Niculescu-Duvaz et al., 2009, “Aryl-quinolyl compounds and their     use”, international patent application publication number WO     2009/130487 A1 published 29 Oct. 2009. -   Solit et al., 2006, “BRAF mutation predicts sensitivity to MEK     inhibition”, Nature, Vol. 439, pp. 358-362. -   Springer et al., 2009, “Pyrido[2,3-b]pyrazine-8-substituted     compounds and their use”, international patent application     publication number WO 2009/077766 A1 published 25 Jun. 2009. -   Wellbrock et al., 2004, “The RAF proteins take centre stage”, Nature     Reviews Molecular Cell Biology, Vol. 5, pp. 875-885. -   Young et al., 2009, “Ras signaling and therapies”, Adv. Cancer Res.,     Vol. 102, pp. 1-17. 

The invention claimed is:
 1. A method of inhibiting the progress of, reducing the rate of progress of or ameliorating a mutant RAS cancer, comprising administering to a subject in need of treatment a therapeutically-effective amount of a compound selected from compounds of the following formula, and pharmaceutically acceptable salts thereof:

wherein -J- is independently:

and wherein —R is independently: —H, -Me, —F, —Cl, —Br, or —I; and wherein —R is positioned meta- or para- on the phenyl ring.
 2. A method according to claim 1, wherein the mutant RAS cancer is: mutant RAS melanoma; mutant RAS lung cancer; mutant RAS colorectal cancer; mutant RAS pancreatic cancer; mutant RAS thyroid cancer; mutant RAS seminoma; mutant RAS myelodisplastic syndrome; mutant RAS liver cancer; mutant RAS leukemia; mutant RAS sarcoma; mutant RAS lymphoma; mutant RAS gastrointestinal cancer; mutant RAS bladder cancer; mutant RAS kidney cancer; mutant RAS ovarian cancer; mutant RAS bile duct cancer; mutant RAS cervix carcinoma; mutant RAS glioma; mutant RAS breast cancer; mutant RAS endometrial cancer; mutant RAS stomach cancer; mutant RAS head and neck cancer; mutant RAS oesophagus carcinoma; or mutant RAS prostate cancer.
 3. A method according to claim 1, wherein the mutant RAS cancer is: mutant RAS melanoma; mutant RAS lung cancer; mutant RAS colorectal cancer; mutant RAS pancreatic cancer; mutant RAS thyroid cancer; mutant RAS seminoma; mutant RAS myelodisplastic syndrome; mutant RAS liver cancer; mutant RAS leukemia; mutant RAS sarcoma; mutant RAS lymphoma; mutant RAS gastrointestinal cancer; mutant RAS bladder cancer; mutant RAS kidney cancer; mutant RAS ovarian cancer; mutant RAS bile duct cancer; or mutant RAS cervix carcinoma.
 4. A method according to claim 1, wherein the mutant RAS cancer is: mutant RAS melanoma; mutant RAS lung cancer; mutant RAS colorectal cancer; mutant RAS pancreatic cancer; or mutant RAS thyroid cancer.
 5. A method according to claim 4, wherein the compound is selected from compounds of the following formula, and pharmaceutically acceptable salts thereof:

wherein —R^(A) is independently —H, -Me, —F, —Cl, —Br, or —I; and wherein —R^(A) is positioned meta- or para- on the phenyl ring.
 6. A method according to claim 5, wherein —R^(A) is —H.
 7. A method according to claim 5, wherein —R^(A) is -Me.
 8. A method according to claim 5, wherein —R^(A) is —F.
 9. A method according to claim 5, wherein —R^(A) is —Cl.
 10. A method according to claim 5, wherein the compound is selected from compounds of the following formula, and pharmaceutically acceptable salts thereof:

wherein —R^(A) is independently —H, -Me, —F, —Cl, —Br, or —I.
 11. A method according to claim 10, wherein —R^(A) is independently —H, -Me, —F, or —Cl.
 12. A method according to claim 5, wherein the compound is selected from compounds of the following formula, and pharmaceutically acceptable salts, hydrates, and solvates thereof:

wherein —R^(A) is independently —H, -Me, —F, —Cl, —Br, or —I.
 13. A method according to claim 12, wherein —R^(A) is independently —H, -Me, —F, or —Cl.
 14. A method according to claim 5, wherein the compound is selected from the following compound and pharmaceutically acceptable salts thereof:


15. A method according to claim 5, wherein the compound is selected from the following compound and pharmaceutically acceptable salts thereof:


16. A method according to claim 5, wherein the compound is selected from the following compound and pharmaceutically acceptable salts thereof:


17. A method according to claim 5, wherein the compound is selected from the following compound and pharmaceutically acceptable salts thereof:


18. A method according to claim 4, wherein the compound is selected from compounds of the following formula, and pharmaceutically acceptable salts thereof:

wherein —R^(B) is independently —H, -Me, —F, —Cl, —Br, or —I; and wherein —R^(B) is positioned meta- or para- on the phenyl ring.
 19. A method according to claim 18, wherein —R^(B) is independently —H, -Me, —F, or —Cl.
 20. A method according to claim 18, wherein —R^(B) is —H.
 21. A method according to claim 18, wherein —R^(B) is -Me.
 22. A method according to claim 18, wherein —R^(B) is —F.
 23. A method according to claim 18, wherein —R^(B) is —Cl.
 24. A method according to claim 18, wherein the compound is selected from compounds of the following formula, and pharmaceutically acceptable salts thereof:

wherein —R^(B) is independently —H, -Me, —F, —Cl, —Br, or —I.
 25. A method according to claim 24, wherein —R^(B) is independently —H, -Me, —F, or —Cl.
 26. A method according to claim 18, wherein the compound is selected from compounds of the following formula, and pharmaceutically acceptable salts thereof:

wherein —R^(B) is independently —H, -Me, —F, —Cl, —Br, or —I.
 27. A method according to claim 26, wherein —R^(B) is independently —H, -Me, —F, or —Cl.
 28. A method according to claim 18, wherein the compound is selected from the following compound, and pharmaceutically acceptable salts thereof:


29. A method according to claim 18, wherein the compound is selected from the following compound, and pharmaceutically acceptable salts thereof:


30. A method according to claim 18, wherein the compound is selected from the following compound, and pharmaceutically acceptable salts thereof: 